Systems and methods for particle multiplexing in droplets

ABSTRACT

Described herein are systems and methods for multiplexed analysis of two or more targets in a test sample including a first set of particles including a first set of target-specific reagents and a first optically detectable identifier capable of emitting a first wavelength indicative of a first target, and at least one second set of particles including a second set of target-specific reagents and a second optically detectable identifier capable of emitting a second wavelength indicative of a second target; and at least one optically detectable reporter probe capable of constitutively emitting a third wavelength in response to reaction of the first set of target-specific reagents with the first target in the test sample and/or reaction of the second set of target-specific reagents with the second target in the test sample, wherein the first wavelength, the second wavelength, and the third wavelength are optically discernable from one another.

If an Application Data Sheet (ADS) has been filed on the filing date ofthis application, it is incorporated by reference herein. Anyapplications claimed on the ADS for priority under 35 U.S.C. § § 119,120, 121, or 365(c), and any and all parent, grandparent,great-grandparent, etc. applications of such applications, are alsoincorporated by reference, including any priority claims made in thoseapplications and any material incorporated by reference, to the extentsuch subject matter is not inconsistent herewith.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of nonprovisional U.S. patentapplication Ser. No. 16/184,397, filed Nov. 8, 2018, titled SYSTEMS ANDMETHODS FOR PARTICLE MULTIPLEXING IN DROPLETS, and naming inventorsSamantha A. Byrnes, Kevin Paul Flood Nichols, and Bernhard Hans Weigl.

All subject matter of the Priority Applications and of any and allapplications related to the Priority Applications by priority claims(directly or indirectly), including any priority claims made and subjectmatter incorporated by reference therein as of the filing date of theinstant application, is incorporated herein by reference to the extentsuch subject matter is not inconsistent herewith.

SUMMARY

In an aspect, a system for multiplexed detection of two or more nucleicacid sequences in a test sample includes, but is not limited to, two ormore sets of particles including a first set of particles, each particleof the first set of particles degradable in response to a firstenvironmental condition and having associated therewith a firstamplification primer set and a first optically detectable identifiercapable of emitting a first wavelength, the first amplification primerset selected to specifically interact with a first nucleic acidsequence, and the first optically detectable identifier indicative ofthe first amplification primer set; and at least one second set ofparticles, each particle of the at least one second set of particlesdegradable in response to a second environmental condition and havingassociated therewith a second amplification primer set and a secondoptically detectable identifier capable of emitting a second wavelength,the second amplification primer set selected to specifically interactwith a second nucleic acid sequence, and the second optically detectableidentifier indicative of the second amplification primer set; and atleast one optically detectable reporter probe capable of constitutivelyemitting a third wavelength in response to amplification of the firstnucleic acid sequence in the test sample and/or the second nucleic acidsequence in the test sample. In addition to the foregoing, other systemaspects are described in the claims, drawings, and text forming a partof the present disclosure.

In an aspect, a system for multiplexed analysis of two or more targetsin a test sample includes, but is not limited to, two or more sets ofparticles including a first set of particles, each particle of the firstset of particles degradable in response to a first environmentalcondition and having associated therewith a first set of one or moretarget-specific reagents and a first optically detectable identifiercapable of emitting a first wavelength, the first set of one or moretarget-specific reagents selected to specifically interact with a firsttarget, and the first optically detectable identifier indicative of thefirst set of one or more target-specific reagents; and at least onesecond set of particles, each particle of the at least one second set ofparticles degradable in response to a second environmental condition andhaving associated therewith a second set of one or more target-specificreagents and a second optically detectable identifier capable ofemitting a second wavelength, the second set of one or moretarget-specific reagents selected to specifically interact with a secondtarget, and the second optically detectable identifier indicative of thesecond set of one or more target-specific reagents; and at least oneoptically detectable reporter probe capable of constitutively emitting athird wavelength in response to a reaction of the first set of one ormore target-specific reagents with the first target in the test sampleand/or a reaction of the second set of one or more target-specificreagents with the second target in the test sample.

In an aspect, a system for multiplexed detection of two or morebacterial nucleic acid sequences in a test sample includes, but is notlimited to, two or more sets of particles including a first set ofparticles, each particle of the first set of particles degradable inresponse to a first temperature condition and having associatedtherewith a first amplification primer set and a first fluorescentidentifier capable of emitting at a first wavelength, the firstamplification primer set selected to specifically interact with a firstbacterial nucleic acid sequence, and the first fluorescent identifierindicative of the first amplification primer set; at least one secondset of particles, each particle of the at least one second set ofparticles degradable in response to a second temperature condition andhaving associated therewith a second amplification primer set and asecond fluorescent identifier capable of emitting at a secondwavelength, the second amplification primer set selected to specificallyinteract with a second bacterial nucleic acid sequence, and the secondfluorescent identifier indicative of the second amplification primerset; and at least one fluorescent intercalating agent capable ofconstitutively emitting at a third wavelength in response toamplification of the first bacterial nucleic acid sequence in the testsample and/or the second bacterial nucleic acid sequence in the testsample.

In an aspect, a system for multiplexed analysis of antibiotic resistancein a bacterial sample includes, but is not limited to, two or more setsof particles including a first set of particles, each particle of thefirst set of particles degradable in response to a first environmentalcondition and having associated therewith a first antibiotic and a firstoptically detectable identifier capable of emitting a first wavelength,the first antibiotic having at least one of bactericidal orbacteriostatic activity against a first subset of bacteria, and thefirst optically detectable identifier indicative of the firstantibiotic; and at least one second set of particles, each particle ofthe at least one second set of particles degradable in response to asecond environmental condition and having associated therewith a secondantibiotic and a second optically detectable identifier capable ofemitting a second wavelength, the second antibiotic having at least oneof bactericidal or bacteriostatic activity against a second subset ofbacteria, and the second optically detectable identifier indicative ofthe second antibiotic; and at least one optically detectable reporterprobe capable of constitutively emitting a third wavelength in responseto viability of bacteria in the bacterial sample. In addition to theforegoing, other system aspects are described in the claims, drawings,and text forming a part of the present disclosure.

In an aspect, a system for multiplexed analysis of two or more antigensin a test sample includes, but is not limited to, two or more sets ofparticles including a first set of particles, each particle of the firstset of particles degradable in response to a first environmentalcondition and having associated therewith a first antibody set and afirst optically detectable identifier capable of emitting a firstwavelength, the first antibody set including two or more antibodiesspecific for proximal targets on a first antigen, wherein the two ormore antibodies of the first antibody set include modifications capableof interacting in an antibody-based proximity assay, and the firstoptically detectable identifier indicative of the first antibody set;and at least one second set of particles, each particle of the at leastone second set of particles degradable in response to a secondenvironmental condition and having associated therewith a secondantibody set and a second optically detectable identifier capable ofemitting a second wavelength, the second antibody set including two ormore antibodies specific for proximal targets on a second antigen,wherein the two or more second antibodies of the second antibody setinclude modifications capable of interacting in an antibody-basedproximity assay, and the second optically detectable identifierindicative of the second antibody set; and at least one opticallydetectable reporter probe capable of constitutively emitting a thirdwavelength in response to the two or more antibodies of the firstantibody set binding to their proximal targets on the first antigenand/or the two or more antibodies of the second antibody set binding totheir proximal targets on the second antigen.

In an aspect, a method for multiplexed analysis of two or more targetsin a test sample includes, but is not limited to, combining in anaqueous medium the test sample, one or more reaction reagents, a firstset of particles, at least one second set of particles, and at least oneoptically detectable reporter probe, wherein each particle of the firstset of particles is degradable in response to a first environmentalcondition and has associated therewith a first set of one or moretarget-specific reagents specific for a first target and a firstoptically detectable identifier capable of emitting a first wavelengthindicative of the first set of one or more target-specific reagents,wherein each particle of the at least one second set of particles isdegradable in response to a second environmental condition and hasassociated therewith a second set of one or more target-specificreagents specific for a second target and a second optically detectableidentifier emitting a second wavelength indicative of the second set ofone or more target-specific reagents, and wherein the at least oneoptically detectable reporter probe constitutively emits a thirdwavelength in response to reaction of the first set of one or moretarget-specific reagents with the first target in the test sample and/orto reaction of the second set of one or more target-specific reagentswith the second target in the test sample; forming a plurality ofreaction droplets by adding an immiscible carrier fluid to the aqueousmedium; performing a reaction with the plurality of formed reactiondroplets; interrogating at least a portion of the plurality of formedreaction droplets for emission of the first wavelength indicative of thefirst set of one or more target-specific reagents, the second wavelengthindicative of the second set of one or more target-specific reagents,and the third wavelength indicative of constitutive emission from the atleast one optically detectable reporter probe; reporting a number of theformed reaction droplets emitting both the first wavelength and thethird wavelength; and reporting a number of the formed reaction dropletsemitting both the second wavelength and the third wavelength. Inaddition to the foregoing, other method aspects are described in theclaims, drawings, and text forming a part of the present disclosure.

In an aspect, a method for multiplexed analysis of two or more nucleicacid sequence targets in a test sample includes, but is not limited to,combining in an aqueous medium the test sample, one or more reactionreagents, a first set of particles, at least one second set ofparticles, and at least one optically detectable reporter probe, whereineach particle of the first set of particles is degradable in response toa first environmental condition and has associated therewith a firstamplification primer set selected to react with a first nucleic acidsequence and a first optically detectable identifier capable of emittinga first wavelength indicative of the first amplification primer set,wherein each particle of the at least one second set of particles isdegradable in response to a second environmental condition and hasassociated therewith a second amplification primer set selected to reactwith a second nucleic acid sequence and a second optically detectableidentifier emitting a second wavelength indicative of the secondamplification primer set, and wherein the at least one opticallydetectable reporter probe is capable of constitutively emitting a thirdwavelength in response to amplification of the first nucleic acidsequence and/or the second nucleic acid sequence; forming a plurality ofreaction droplets by adding an immiscible carrier fluid to the aqueousmedium; performing an amplification reaction with the plurality offormed reaction droplets; interrogating at least a portion of theplurality of formed reaction droplets for emission of the firstwavelength indicative of the first amplification primer set, the secondwavelength indicative of the second amplification primer set, and thethird wavelength indicative of constitutive emission from the at leastone optically detectable reporter probe in response to amplification ofthe first and/or the second nucleic acid sequence in the formed reactiondroplets; reporting a number of the formed reaction droplets emittingboth the first wavelength and the third wavelength; reporting a numberof the formed reaction droplets emitting both the second wavelength andthe third wavelength. In addition to the foregoing, other method aspectsare described in the claims, drawings, and text forming a part of thepresent disclosure.

In an aspect, a method for multiplexed analysis of antibiotic resistancein a bacterial sample includes, but is not limited to, combining in anaqueous medium the test sample, one or more reaction reagents, a firstset of particles, at least one second set of particles, and at least oneoptically detectable reporter probe, wherein each particle of the firstset of particles is degradable in response to a first environmentalcondition and has associated therewith a first antibiotic having atleast one of bactericidal or bacteriostatic activity against a firstsubset of bacteria and a first optically detectable identifier capableof emitting a first wavelength indicative of the first antibiotic,wherein each particle of the at least one second set of particles isdegradable in response to a second environmental condition and hasassociated therewith a second antibiotic having at least one ofbactericidal or bacteriostatic activity against a second subset ofbacteria and a second optically detectable identifier capable ofemitting a second wavelength indicative of the second antibiotic, andwherein the at least one optically detectable reporter probe is capableof constitutively emitting a third wavelength in response to viabilityof bacteria in the bacterial sample; forming a plurality of reactiondroplets by adding an immiscible carrier fluid to the aqueous medium;performing a reaction with the plurality of formed reaction droplets;interrogating at least a portion of the plurality of formed reactiondroplets for emission of the first wavelength indicative of the firstantibiotic, the second wavelength indicative of the second antibiotic,and the third wavelength indicative of constitutive emission from the atleast one optically detectable reporter probe in response to viabilityof bacteria in the bacterial sample; reporting a number of the formedreaction droplets emitting both the first wavelength and the thirdwavelength; and reporting a number of the formed reaction dropletsemitting both the second wavelength and the third wavelength. Inaddition to the foregoing, other method aspects are described in theclaims, drawings, and text forming a part of the present disclosure.

In an aspect, a method for multiplexed analysis of two or more antigensin a test sample includes, but is not limited to combining in an aqueousmedium the test sample, one or more reaction reagents, a first set ofparticles, at least one second set of particles, and at least oneoptically detectable reporter probe, wherein each particle of the firstset of particles is degradable in response to a first environmentalcondition and has associated therewith a first antibody set and a firstoptically detectable identifier capable of emitting a first wavelength,the first antibody set including two or more antibodies specific forproximal targets on a first antigen, the two or more antibodies of thefirst antibody set including modifications capable of interacting in anantibody-based proximity assay, and the first optically detectableidentifier indicative of the first antibody set, wherein each particleof the second set of particles is degradable in response to a secondenvironmental condition and has associated therewith a second antibodyset and a second optically detectable identifier capable of emitting asecond wavelength, the second antibody set including two or moreantibodies specific for proximal targets on a second antigen, the two ormore antibodies of the second antibody set including modificationscapable of interacting in an antibody-based proximity assay, and thesecond optically detectable identifier indicative of the second antibodyset, and wherein the at least one optically detectable reporter probe iscapable of constitutively emitting a third wavelength in response to thetwo or more antibodies of the first antibody set binding to theirproximal targets on the first antigen and/or the two or more antibodiesof the second antibody set binding to their proximal targets on thesecond antigen; forming a plurality of reaction droplets by adding animmiscible carrier fluid to the aqueous medium; performing a reactionwith the plurality of formed reaction droplets; interrogating at least aportion of the plurality of formed reaction droplets for emission of thefirst wavelength indicative of the first antibody set, the secondwavelength indicative of the second antibody set, and the thirdwavelength indicative of constitutive emission from the at least oneoptically detectable reporter probe in response to the two or moreantibodies of the first antibody set binding to the first antigen and/orthe two or more antibodies of the second antibody set binding to thesecond antigen; reporting a number of the formed reaction dropletsemitting both the first wavelength and the third wavelength; andreporting a number of the formed reaction droplets emitting both thesecond wavelength and the third wavelength.

The foregoing summary is illustrative only and is not intended to be inany way limiting. In addition to the illustrative aspects, embodiments,and features described above, further aspects, embodiments, and featureswill become apparent by reference to the drawings and the followingdetailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic of a system for multiplexed analysis of two ormore targets in a test sample including two or more sets of particlesand at least one optically detectable reporter probe.

FIG. 2 illustrates a method for multiplexed analysis using a system suchas shown in FIG. 1 .

FIG. 3 is a schematic of a system for multiplexed analysis of two ormore nucleic acid sequences in a test sample including two or more setsof particles and at least one optically detectable reporter probe.

FIG. 4 is a schematic of a system for multiplexed analysis of two ormore bacterial nucleic acid sequences in a test sample including two ormore sets of particles and at least one fluorescent intercalating agent.

FIG. 5 is a schematic of a system for multiplexed analysis of antibioticresistance in a bacterial sample including two or more sets of particlesand at least one optically detectable reporter probe.

FIG. 6 is a schematic of a system for multiplexed analysis of two ormore antigens in a test sample including two or more sets of particlesand at least one optically detectable reporter probe.

FIG. 7 is a block diagram of a method for multiplexed analysis of two ormore targets in a test sample using a system such as shown in FIG. 1 .

FIG. 8 is a block diagram of a method for multiplexed analysis of two ormore nucleic acid sequences in a test sample using a system such asshown in FIG. 3 .

FIG. 9 is a block diagram of a method for multiplexed analysis ofantibiotic resistance in a bacterial sample using a system such as shownin FIG. 5 .

FIG. 10 is a block diagram of a method for multiplexed analysis of twoor more antigens in a test sample using a system such as shown in FIG. 6.

FIG. 11A shows a brightfield microscope image of formed agaroseparticles.

FIG. 11B shows a composite of a brightfield microscope image of FIG. 11Aoverlaid with a fluorescent image of the same field.

FIG. 11C shows a brightfield microscope image of formed agaroseparticles washed in water.

FIG. 11D shows a composite of the brightfield microscope image of FIG.11C overlaid with a fluorescent image of the same field.

FIG. 12 shows a fluorescence microscope image at 200× magnification ofthe formed agarose particles with quantum dots.

FIG. 13 shows a microscope image at 200× magnification of the formedreaction droplets post PCR amplification.

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying drawings, which form a part hereof. In the drawings,similar symbols typically identify similar components, unless contextdictates otherwise. The illustrative embodiments described in thedetailed description, drawings, and claims are not meant to be limiting.Other embodiments may be utilized, and other changes may be made,without departing from the spirit or scope of the subject matterpresented here.

Described herein are systems and methods for multiplexed analysis of twoor more targets in a sample using two or more sets of particles and areporter probe. The two or more sets of particles and the reporter probeare designed for use in multiplexed reactions of a test sample inaqueous-in-oil reaction droplets. The particles include target-specificreagents (e.g., target-specific amplification primers) and an identifiermatched to the target-specific reagents and hence a specific target(e.g., a specific target nucleic acid sequence) while the reporter probeis a constitutive reporter of a reaction type (e.g., amplification). Theparticles, reporter probe, test sample and other reaction reagents canbe stochastically distributed into aqueous-in-oil reaction droplets anda reaction(s) performed. Each reaction droplet is assessed for a signalfrom the identifier (e.g., a first fluorescent color) matched to thetarget-specific reagents (and to the specific target) and a signal fromthe constitutive reporter of the reaction type (e.g., a secondfluorescent color) indicating that a reaction has occurred (e.g.,amplification). A reaction droplet emitting signals from both theidentifier (e.g., a first fluorescent color) and the reporter probe(e.g., a second fluorescent color) indicates that the reaction with thespecific target (e.g., amplification of a specific target nucleic acidsequence) has occurred. In this way, multiple targets can be assessed ina single multiplexed analysis.

With reference to FIG. 1 , shown is a non-limiting example of a systemfor multiplexed analysis of two or more targets in a test sample whichcan serve as a context for one or more systems and/or methods describedherein. System 100 includes two or more sets of particles 105 and atleast one optically detectable reporter probe 130. The two or more setsof particles 105 include a first set of particles 110 and at least onesecond set of particles 120. Particles 112 a, 112 b, 112 c, 112 d, 112e, and 112 f are representative of particles in the first set ofparticles 110 and are degradable in response to a first environmentalcondition. Each of the particles 112 a-112f include a first set of oneor more target-specific reagents 114 and a first optically detectableidentifier 116. The first set of one or more target-specific reagents114 are selected to specifically interact with a first target in thesample. The first optically detectable identifier 116 is capable ofemitting a first wavelength (represented by the vertical lines)indicative of the first set of one or more target-specific reagents 114.The first optically detectable identifier 116 is further indicative ofthe first target to which the first set of one or more target-specificreagents 114 is capable of reacting with in a test sample. In general,the optically detectable identifier indicates that a particular set ofone or more target-specific reagents are present in a given set ofparticles. In an aspect, the optically detectable identifier acts as abarcode for the set of one or more target-specific reagents and thespecific target.

Particles 122 a, 122 b, 122 c, 122 d, 122 e, and 122 f arerepresentative of particles in the second set of particles 120 and aredegradable in response to a second environmental condition. In someembodiments, the second environmental condition is the same as the firstenvironmental condition (e.g., the same temperature or pH condition). Insome embodiments, the second environmental condition differs from thefirst environmental condition (e.g., differing temperature or pHconditions). Each of the particles 122 a-122f include a second set ofone or more target-specific reagents 124 and a second opticallydetectable identifier 126. The second set of one or more target-specificreagents 124 are selected to specifically interact with a second targetin the sample. The second optically detectable identifier 126 is capableof emitting a second wavelength (represented by diagonal lines)indicative of the second set of one or more target-specific reagents124. The second optically detectable identifier 126 is furtherindicative of the second target to which the second set of one or moretarget-specific reagents 124 is capable of reacting with in a testsample.

System 100 further includes at least one optically detectable reporterprobe 130 capable of constitutively (as shown by arrow 132) emitting athird wavelength 134 (represented by the cross-hatched lines) inresponse to reaction of the first set of one or more target-specificreagents 114 with the first target in the sample and/or reaction of thesecond set of one or more target-specific reagents 124 with the secondtarget in the sample. The optically detectable reporter probeconstitutively emits that third wavelength in response to detecting areaction. In some embodiments, the reaction detected by the opticallydetectable reporter probe only occurs if both the set of target-specificreagents and the relevant specific target are present in a givenreaction droplet.

With reference to FIG. 2 , shown is a non-limiting example of a methodfor multiplexed analysis of two or more targets in a test sample usingtwo or more sets of particles and an optically detectable reporter probesuch as described in the system of FIG. 1 . Method 200 includes at step205 combining the components of system 100 with a test sample andreaction reagents in an aqueous medium. System 100 includes two or moresets of particles 105 including a first set of particles 110(represented by the circles with vertical lines) and at least one secondset of particles 120 (represented by the circles with diagonal lines).System 100 further includes at least one optically detectable reporterprobe 130. Method 200 includes at step 210 adding an immiscible carrierfluid (e.g., oil with surfactant) to the aqueous medium and at step 215forming reaction droplets 220. Reaction droplets 220 are aqueous-in-oildroplets that can be formed by bulk emulsion (e.g., shaking orvortexing) or with a microfluidic device. The particles of the first setof particles 110, the particles of the at least one second set ofparticles 120, the optically detectable reporter probe 130, and anytargets (shown as Target 1 and Target 2) within the test sample added atstep 205 are stochastically distributed into the formed reactiondroplets 220 at step 215. The reaction droplets 220 can be ofmono-disperse or poly-disperse sizes. At step 225, method 200 includesperforming a reaction with the formed reaction droplets 220 (e.g., anamplification reaction). Following completion of the reaction with theformed reaction droplets 220, method 200 includes at step 230interrogating the formed reaction droplets 220 for a first wavelength235 (depicted as vertical lines) indicative of the first set of one ormore target-specific reagents associated with the first set of particles110, a second wavelength 240 (depicted as diagonal lines) indicative ofthe second set of one or more target-specific reagents associated withthe at least one second set of particles 120, and at step 250interrogating the formed reaction droplets 220 for a third wavelength255 (depicted as cross-hatching) constitutively emitted from theoptically detectable reporter probe in response to at least one of thefirst set of target-specific reagents reacting with a first target(Target 1) in the test sample and/or the second set of target-specificreagents reactive with a second target (Target 2) in the test sample.Method 200 further includes at step 260 reporting a number of reactiondroplets 220 emitting the first wavelength 235 and the third wavelength255 and at step 265 reporting a number of reaction droplets 220 emittingthe second wavelength 240 and the third wavelength 255. Reactiondroplets emitting both the third wavelength and the first or secondwavelength are indicative of the presence of the desired target (e.g., aspecific nucleic acid sequence) and a generic reaction having beenperformed on/with the desired target (e.g., amplification).

Systems and methods are described herein for multiplexed analysis of twoor more targets in a test sample. The two or more targets can includetwo or more specific targets in the test sample. The two or more targetscan include two or more specific analytes in the test sample. In someembodiments, the two or more targets in the test sample include two ormore nucleic acid sequences. For example, the two or more targets caninclude two or more specific DNA, RNA, or oligonucleotide sequences in atest sample. The two or more targets can include two or more specificcell types. For example, the two or more targets can include two or moretypes of bacteria or immune cells. The two or more targets can includetwo or more specific antigens.

The first set of one or more target-specific reagents is selected tospecifically interact with a first target while the second set of one ormore target-specific reagents is selected to specifically interact witha second target. For example, the one or more target specific reagentscan include a sequence (e.g., a nuclei acid or amino acid sequence), athree dimensional structure, or an affinity that enables them tospecifically interact with a target in a sample. In an aspect, the firstset of one or more target-specific reagents is selected to specificallyinteract with a first nucleic acid sequence and the second set of one ormore target-specific reagents selected to specifically interact with asecond nucleic acid sequence. In some embodiments, the two or moretargets in the test sample comprise two or more types of bacteria in abacterial sample. For example, the two or more targets in the testsample can include two or more types of bacteria associated with sepsisor tuberculosis. In an aspect, the first set of one or moretarget-specific reagents is selected to specifically interact with afirst subset of bacteria and the second set of one or moretarget-specific reagents is selected to specifically interact with asecond subset of bacteria. In some embodiments, the two or more targetsin the test sample include two or more binding targets. In someembodiments, the two or more targets in the test sample include two ormore antigens in the test sample. For example, the two or more targetscan include two or more antigens free in solution. For example, the twoor more targets can include two or more antigens associated with one ormore cell types, e.g., one or more types of immune or inflammatorycells. In an aspect, the first set of one or more target-specificreagents is selected to specifically interact with a first antigen andthe second set of one or more target-specific reagents selected tospecifically interact with a second antigen. In an aspect, each set ofone or more target-specific reagents is selected to specificallyinteract with at least one protein, peptide, carbohydrate, lipid, ornucleic acid sequence. Other non-limiting examples of targets includeantigens, receptors, cell surface markers, small molecule compounds,organic compounds, or inorganic compounds. In some embodiments, the twoor more targets can include two or more targets (e.g., DNA, RNA,protein, carbohydrate, lipid, and the like) associated with a specificcell type(s) (e.g., blood, body fluid, or tissue cells, bacteria, fungi,parasites, plant cells, and the like).

Systems and methods are described herein for multiplexed analysis of twoor more targets in a test sample. In an aspect, the test sample is acomplicated or complex test sample, including many components (e.g., abiological sample such as blood or urine; or an environmental samplesuch as water from a well or other drinking water source). In an aspect,the test sample is a simple or defined test sample, including just twoor more components. In an aspect, the test sample includes a bodilyfluid (e.g., blood, urine, lymph, sputum, cerebrospinal fluid, semen,saliva, synovial fluid, mucus, amniotic fluid, vaginal secretions,breast milk, bile, aqueous humor, gastric acid, pus, phlegm, feces, orother bodily fluid or secretion), a tissue sample (e.g., a biopsysample), or a swab sample (e.g., a swab sample taken from a surface of abody or body part). In some embodiments, the test sample includes anenvironmental sample (e.g., a sample of water, air, soil, plant, asurface, food, beverage, medicine, and the like). In an aspect, the testsample is a defined sample including a defined population of targets.For example, the test sample can be formulated to contain a definednumber and set of targets for use in the multiplexed analysis.

System 100 includes two or more sets of particles 105 including a firstset of particles 110 and at least one second set of particles 120,wherein each particle of the first set of particles 110 is degradable inresponse to a first environmental condition and each particle of the atleast one second set of particles 120 is degradable in response to asecond environmental condition. In an aspect, degradable in response tothe first environmental condition and/or the second environmentalcondition comprises at least one of melt, liquefy, disperse, dissolve,decompose, disintegrate, break apart, deform, or change phase inresponse to the first environmental condition and/or the secondenvironmental condition. In an aspect, degradable in response to thefirst environmental condition and/or the second environmental conditioncomprises at least partially melting, liquefying, dispersing,dissolving, decomposing, disintegrating, breaking apart, deforming, orchanging phase in response to the first environmental condition and/orthe second environmental condition. In some embodiments, each particleis capable of releasing or dispersing the set of one or moretarget-specific reagents and/or the optically detectable identifier inresponse to the environmental condition. In some embodiments, eachparticle degrades sufficiently to release or disperse the set of one ormore target-specific reagents and/or the optically detectableidentifier, but does not completely disintegrate. For example, theporosity of each particle may be altered in response to theenvironmental condition, allowing for release or dispersal of thetarget-specific reagents and/or the optically detectable identifier fromthe particle. For example, each particle can at least one of shrink orswell in response to an environmental condition.

In an aspect, the first environmental condition and the secondenvironmental condition are identical environmental conditions. Forexample, the first environmental condition can be equivalent to or thesame as the second environmental condition. For example, the particlesin the first set of particles 110 and the particles in the at least onesecond set of particles 120 can degrade (e.g., melt, liquefy, disperse,dissolve, decompose, disintegrate, break apart, deform, or change phase)at the same temperature. In an aspect, the first environmental conditionand the second environmental condition are different environmentalconditions. For example, the particles in the first set of particles 110can degrade at a first temperature and the particles in the at least onesecond set of particles 120 can degrade at a second temperature, whereinthe first and second temperatures are different. In an aspect, the firstenvironmental condition and the second environmental condition compriseat least one of temperature, pH, chemical reaction, electric field, orelectromagnetic energy.

In an aspect, the particles of the first set of particles 110 and of theat least one second set of particles 120 include a structure formed froma degradable material that is degradable in response to at least thefirst environmental condition or the second environmental condition. Inan aspect, each particle of the first set of particles 110 is formedfrom a first material that degrades in response to a first environmentalcondition and each particle of the at least one second set of particles120 is formed from a second material that degrades in response to asecond environmental condition. In an aspect, the particles of the firstset of particles 110 and of the at least one second set of particles 120are formed from the same material that degrades in response to the firstenvironmental condition and/or the second environmental condition. In anaspect, each particle of the first set of particles 110 and the at leastone second set of particles 120 is formed from a material that at leastone of melts, liquefies, disperses, dissolves, decomposes,disintegrates, breaks apart, deforms, or changes phase in response to afirst environmental condition and/or a second environmental condition.In some embodiments, each particle of the first set of particles 110 andthe at least one second set of particles 120 is formed from a materialthat releases or disperses the set of one or more target-specificreagents and/or the optically detectable identifier in response to theenvironmental condition. In some embodiments, each particle of the firstset of particles 110 and the at least one second set of particles 120 isformed from a material that degrades sufficiently to release or dispersethe set of one or more target-specific reagents and/or the opticallydetectable identifier, but does not completely disintegrate. Forexample, the particles may be formed from a material (e.g., a hydrogel)which has increased porosity in response to an environmental condition,allowing for release or dispersal of the target-specific reagents and/orthe optically detectable identifier from the particles. For example, theparticles can be formed from a material that at least one of shrinks orswells in response to an environmental condition, allowing for releaseor dispersal of the target-specific reagents and/or the opticallydetectable identifier from the particles.

In some embodiments, system 100 can include more than two sets ofparticles, wherein each set of particles includes a unique set oftarget-specific reagents selected to interact with one of many potentialtargets in a test sample and a unique optically detectable identifierindicative of the one of many potential targets in the sample. In anaspect, the number of unique sets of particles range from three sets ofparticles to twenty sets of particles. In an aspect, system 100 includesthree, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, ortwenty sets of particles, each set of particles including a set of oneor more target-specific reagents and an optically detectable identifier.

In an aspect, system 100 includes at least one third set of particles,each particle of the at least one third set of particles degradable inresponse to at least one of the first, the second, or a thirdenvironmental condition and having associated therewith a third set ofone or more target-specific reagents and a third optically detectableidentifier capable of emitting a fourth wavelength, the third set of oneor more target-specific reagents selected to specifically interact witha third target in the sample, and the third optically detectableidentifier indicative of the third set of one or more target-specificreagents; and wherein the at least one optically detectable reporterprobe is capable of constitutively emitting the third wavelength inresponse to at least the first set of one or more target-specificreagents reacting with the first target in the sample, the second set ofone or more target-specific reagents reacting with the second target inthe sample, and/or the third set of one or more target-specific reagentsreacting with the third target, and wherein the first wavelength, thesecond wavelength, the third wavelength, and the fourth wavelength areoptically discernable from one another.

In an aspect, the particles of the first set of particles 110 and of theat least one second set of particles 120 include a structure. Forexample, the particles of the first set of particles and of the at leastone second set of particles can include a structure formed from adegradable material that is degradable in response to at least the firstenvironmental condition or the second environmental condition. Forexample, the structure can include a micro- or nano-bead or particleformed from a solid or semi-solid degradable material. In an aspect, thestructure is a bead or particle formed from a solid or semi-solid atleast partially degradable material. In an aspect, the structure isspherical in shape, e.g., a microsphere. However, other non-sphericalshapes are contemplated, e.g., irregular shape, cuboid, and the like. Inan aspect, the particles range in size from about 0.1 micron to about 20microns in diameter. For example, each particle can be about 0.1 micron,0.2 micron, 0.5 micron, 0.75 micron, 1 micron, 2 micron, 3 micron, 4micron, 5 micron, 6 micron, 7 micron, 8 micron, 9 micron, 10 micron, 11micron, 12 micron, 13 micron, 14 micron, 15 micron, 16 micron, 17micron, 18 micron, 19 micron, or 20 micron in diameter. In an aspect,each of the particles has a structure that is smaller thanaqueous-in-oil reaction droplets formed with an immiscible carrier fluidand an aqueous medium containing the two or more sets of particles andthe at least one optically detectable reporter probe.

In an aspect, the particles of the first set of particles 110 and of theat least one second set of particles 120 include a structure formed froma material that is stable in an aqueous environment. For example, eachparticle can be formed from a material that is stable in an aqueousmedium including one or more reaction reagents (e.g., enzymes, ions,buffers, culture medium, and the like) for use in performing themultiplexed analysis. For example, each particle can be formed from amaterial that is stable in an aqueous environment until the contents ofthe particle, i.e., the set of one or more target-specific reagents andthe at least one optically detectable identifier, are released inresponse to an environmental condition into a reaction droplet forperforming the multiplex analysis.

In an aspect, the particles of the first set of particles 110 and of theat least one second set of particles 120 include a structure formed froma degradable gel. The degradable gel can include a colloidal network,polymer network, or solid jelly-like material with a three-dimensionalcross-linked network capable of trapping and containing fluids. Thenetwork structure may result from physical bonds (e.g., ionicinteractions, hydrogen bonds, or hydrophobic interactions) or chemicalbonds (e.g., chemical cross-linking agents).

In an aspect, the particles of the first set of particles 110 and of theat least one second set of particles 120 include a structure formed froma degradable gel that is a hydrogel. The hydrogel can be athree-dimensional cross-linked network of natural and/or synthetichydrophilic polymers with relatively high water content, e.g, up to orover 90% water. In an aspect, the particles are formed from an amorphoushydrogel, a semi-crystalline hydrogel, or a crystalline hydrogel. In anaspect, the particles are formed from a hydrogel formed by linkingpolymer chains via a chemical reaction, ionizing radiation, and/orphysical interactions such as entanglements, electrostatics, orcrystalline formation.

In an aspect, the particles of the first set of particles 110 and of theat least one second set of particles 120 include a structure formed froma neutral or nonionic hydrogel. In an aspect, the particles of the firstset of particles 110 and of the at least one second set of particles 120are formed from an ionic (anionic or cationic) hydrogel. In an aspect,the particles of the first set of particles 110 and of the at least onesecond set of particles 120 are formed from an amphoteric hydrogelcontaining both acidic and basic groups. In an aspect, the particles ofthe first set of particles 110 and of the at least one second set ofparticles 120 are formed from a zwitterionic hydrogel containing bothanionic and cationic groups in repeating units.

In an aspect, the particles of the first set of particles 110 and of theat least one second set of particles 120 include a structure formed froma homopolymeric hydrogel having a polymeric network derived from asingle species of monomer. In an aspect, each particle of the first setof particles 110 and of the at least one second set of particles 120 isformed from a copolymeric hydrogel having two or more different monomerspecies with at least one hydrophilic component and organized in random,block, or alternating configurations along the chain of the polymernetwork. In an aspect the hydrogel includes an interpenetrating polymernetwork formed from two independent cross-linked synthetic or naturalpolymer components. In an aspect, the hydrogel includes asemi-interpenetrating polymer network formed from one component that isa cross-linked polymer and another component that is a non-cross-linkedpolymer. In an aspect, the hydrogel includes a sequentialinterpenetrating or semi-interpenetrating polymer network. In an aspect,the superstructure of the particles is a hydrogel formed by bulk,solution, or suspension polymerization.

In an aspect, the particles of the first set of particles 110 and of theat least one second set of particles 120 include a structure formed froma natural hydrogel material. For example, the particles can be formedfrom natural polymers including proteins, non-limiting examples of whichinclude collagen, gelatin, fibrin, or elastin. For example, theparticles can be formed from natural polymers including polysaccharidessuch as chitosan, starch, alginate, and agarose. For example, theparticles can include methylcellulose, hyaluronic acid, and othernaturally derived polymers.

In an aspect, the particles of the first set of particles 110 and of theat least one second set of particles 120 include a structure formed fromsynthetic polymers. For example, the particles can be formed throughchemical polymerization methods from one or more of polyethylene glycol(PEG), polyvinyl alcohol, sodium polyacrylate, acrylate polymers,acrylamide polymers, methacrylate, acrylonitrile, and derivatives,combinations, and/or salts thereof, and copolymers with an abundance ofhydrophilic groups. Other non-limiting examples include PEG-diacylate(PED-DA), thiol and/or acrylate modified PEG, and azide and/or alkynemodified PEG.

In an aspect, the particles of the first set of particles 110 and of theat least one second set of particles 120 include a structure formed froma form of acrylamide For example, the particles can be formed frompolyacrylamide. For example, the particles can be formed from a polymerof N-isopropylacrylamide, non-limiting examples of which includepoly(N-isopropylacrylamide) (pNIPA, pNIPAAm, pNIPAA or pNIPAm)thermosensitive polymer or copolymer-based hydrogels. Other non-limitingexamples include poly(N-octyl acrylamide), poly(N-tert-butylacrylamide), poly(N-phenyl acrylamide), and poly(N-sec-butylacrylamide).

In an aspect, the particles of the first set of particles 110 and of theat least one second set of particles 120 include a structure formed fromagarose. For example, the particles can be formed from repeatingagarobiose units extracted from red seaweed. For example, particles canbe formed from a linear polymer composed of a disaccharide ofD-galactose and 3,6-anhydro-L-galactopyranose. For example, theparticles can be formed from a linear galactan hydrocolloid isolatedfrom agar or agar-bearing marine algae.

In an aspect, the particles of the first set of particles 110 and of theat least one second set of particles 120 include a structure formed fromlow melt agarose. In an aspect, each particle of the first set ofparticles and the at least one second set of particles is formed from alow melt agarose with a melting temperature of less than or equal toabout 95° C. to less than or equal to about 45° C. For example, the lowmelt agarose can have a melting temperature of less than or equal toabout 95° C., 90° C., 85° C., 80° C., 75° C., 70° C., 65° C., 60° C.,55° C., 50° C., or 45° C. In an aspect, each particle of the first setof particles and the at least one second set of particles is formed froma low melt agarose with a melting temperature less than or equal toabout 65° C. (e.g., product number A9414 from Sigma-Aldrich Corp., St.Louis, Mo.). In an aspect, each particle of the first set of particlesand the at least one second set of particles is formed from an ultralowmelt agarose with a melting temperature less than or equal to about 50°C. (e.g., product number A5030 from Sigma-Aldrich Corp., St. Louis,Mo.).

In an aspect, the particles of the first set of particles 110 and of theat least one second set of particles 120 include a structure formed froma degradable alginate. In an aspect, the alginate is used in the form ofa hydrogel. In an aspect, the alginate is formed from some combinationof (1,4)-linked β-D-mannuronate (M) and a-L-guluronate (G) residues. Thealginate can include linear copolymers containing blocks of M and Gresidues, for example blocks of consecutive G residues, blocks ofconsecutive M residues, and blocks of alternating M and G residues. Inan aspect, percentage of G blocks dictates the physical properties ofthe resultant hydrogel. For example, alginate with a higher percentageof G blocks may have a relatively high stiffness. In an aspect, the M/Gratio dictates the physical properties of the resultant hydrogel. In anaspect, sequence, G-block length, and/or molecular weight dictatephysical properties of the resultant hydrogel. In an aspect, thealginate includes a mixture of high and low molecular weight alginatepolymers. In an aspect, the alginate is sourced from brown algaePhaeophyceae including, but not limited to Laminaria hyperborea,Laminaria digitate, Laminaria japonica, Ascophyllum nodosum, andMacrocystis pyrifera. In an aspect, the alginate is sourced frombacterial biosynthesis. For example, bacterial alginate can be producedvia biosynthesis from Azotobacter or Pseudomonas bacteria. In an aspect,each particle of the first set of particles and the at least one secondset of particles is formed from an alginate salt. For example, thealginate salt can include sodium alginate, potassium alginate, calciumalginate, or a combination thereof.

In an aspect, the particles of the first set of particles 110 and of theat least one second set of particles 120 include a structure formed froma derivative of alginate. For example, particles can be formed from analginate material to which other elements have been added. In someembodiments, each particle of the first set of particles and the atleast one second set of particles is formed by adding hydrophobicmoieties to the alginate backbone. For example, amphiphilic alginatederivatives can be formed by adding long alkyl chains (e.g., dodecyl,octadecyl) to the alginate backbone. Other elements that could be addedto the alginate backbone to generate amphiphilic alginate include, butare not limited to, dodecylamine, cholesterol, poly(ε-caprolactone), andpoly(butyl) methacrylate. In some embodiments, the particles can includecell-interactive alginate to which cell-adhesive peptides have beenchemically added as side-chains to the alginate backbone. For example,the alginate backbone can be modified with peptides including anarginine-glycine-aspartic acid (RGD) sequence.

In an aspect, the particles of the first set of particles 110 and of theat least one second set of particles 120 include a structure formed byionically crosslinking an aqueous alginate solution. In someembodiments, the aqueous alginate solution is cross-linked with an ioniccross-linking agent. For example, an aqueous alginate solution can becross-linked using a divalent or bivalent cation (e.g., Ca²⁺, Mg²⁺,Fe²⁺, or Ni²⁺). For example, an aqueous alginate solution can becross-linked using calcium chloride (CaCl₂). Other non-limiting examplesof ionic cross-linking agents for crosslinking alginate include calciumsulfate and/or calcium carbonate.

In an aspect, the particles of the first set of particles 110 and of theat least one second set of particles 120 include a structure formed bycovalently cross-linking the alginate. In some embodiments, the alginateis cross-linked with a covalent cross-linking agent. For example, thealginate can be cross-linked with poly(acrylamide-co-hydrazide) oradipic acid dihydrazide. In some embodiments, the alginate is photocross-linked. For example, alginate modified with methacrylate can becross-linked by exposure to an argon ion laser. For example, acombination of photosensitive plyallylamine and alginate can becross-linked in response to ultra violet radiation. In an aspect, eachparticle of the first set of particles and the at least one second setof particles is formed with alginate using a combination of an ioniccross-linking agent and a covalent cross-linking agent.

In an aspect, the particles of the first set of particles 110 and of theat least one second set of particles 120 is formed from alginatecombined with thermo-sensitive co-polymers by a thermal process. Forexample, alginate can be cross-linked by UV irradiation in the presenceof N-isopropylacrylamide and poly(ethyleneglycol)-co-poly(ε-caprolactone).

In an aspect, the particles of the first set of particles 110 and of theat least one second set of particles 120 include a structure formed froma phase-change material that works by absorbing and storing/releasingthermal energy. In an aspect, the phase-change material is an organicmaterial, e.g., paraffin, carbohydrate, or lipid derived. In an aspect,the phase-change material is an inorganic material, e.g., salt hydrates.

In an aspect, the particles of the first set of particles 110 and of theat least one second set of particles 120 include a structure formed froma degradable sugar. In an aspect, the degradable sugar is a dissolvablesugar. In an aspect, the degradable sugar is a disaccharide. Forexample, the degradable sugar can be a form of trehalose dihydrate(α-D-glucopyranosyl, α-D-glucopyranoside). In an aspect, the degradablesugar is a polysaccharide (e.g., starch, cellulose, glycogen, chitin,callose, laminarin, chrysolaminarin, xylan, arabinoxylan, mannan,fucoidan, pectins, or galactomannan). In an aspect, the degradable sugaris an oligosaccharide (e.g., raffinose, maltodextrins, orcellodextrins).

In an aspect, the particles of the first set of particles 110 and of theat least one second set of particles 120 include a structure thatincludes a degradable coating. In an aspect, the particles are formedfrom a solid or semi-solid material and further coated with a degradablematerial. In an aspect, the set of one or more target-specific reagentsand the optically detectable identifier are incorporated into andreleasable from a degradable coating in response to an environmentalcondition or change in environmental condition, wherein theenvironmental condition includes at least one of temperature, pH,chemical reaction, enzymatic reaction, electrical field, orelectromagnetic energy.

In an aspect, the particles of the first set of particles 110 and of theat least one second set of particles 120 include a structure formed froma solid matrix. For example, particles can be formed from a solidmatrix, e.g., polystyrene, to which the set of one or moretarget-specific reagents and the optically detectable identifier areassociated with. For example, the set of one or more target-specificreagents and the optically detectable identifier can be attached to thesolid matrix. For example, the set of one or more target-specificreagents and the optically detectable identifier can be included in acoating formed around the surface of the solid matrix. In some aspects,the coating including the set of one or more target-specific reagentsand the optically detectable identifier is degradable allowing releaseof the set of one or more target-specific reagents and the opticallydetectable identifier. In an aspect, each particle is formed from latexand includes a coating degradable in response to an environmentalcondition.

In an aspect, the set of one or more target-specific reagents and theoptically detectable identifier are incorporated into the particlesduring the formation of the particles. For example, the set of one ormore target-specific reagents and the optically detectable identifiercan be incorporated into liquefied low melt agarose prior to gelling. Inan aspect, the set of one or more target-specific reagents and theoptically detectable identifier are incorporated into the particlesafter formation of the particles. For example, the set of one or moretarget-specific reagents and the optically detectable identifier can besoaked, infused, absorbed, adsorbed, diffused, or otherwise integratedinto or onto previously formed particles. In an aspect, the set of oneor more target-specific reagents and the optically detectable identifierare attached to the exterior of the particles. In an aspect, the set ofone or more target-specific reagents and the optically detectableidentifier are incorporated into a coating associated with the exteriorof the particles.

In an aspect, the particles of the first set of particles 110 and the atleast one second set of particles 120 are formed from a porous material.For example, particles can be formed from a porous material throughwhich the set of one or more target-specific reagents, the opticallydetectable identifier, or a combination thereof can freely diffuse froman internal reservoir of the particle. In an aspect, the particles ofthe first set of particles and of the at least one second set ofparticles are hollow microspheres, the center of which forms a reservoirfor the set of one or more target-specific reagents and the opticallydetectable identifier.

In an aspect, the particles of the first set of particles 110 and of theat least one second set of particles 120 include a structure formed froma degradable material that is degradable in response to at least thefirst environmental condition or the second environmental condition. Inan aspect, each of the particles in each of the first set of particlesand the at least one second set of particles is formed from the samedegradable material degradable in response to the same environmentalcondition, e.g., a specific temperature. In an aspect, each of theparticles in the first set of particles is formed from a firstdegradable material degradable in response to a first environmentalcondition and each of the particles in the at least one second set ofparticles is formed from a second degradable material degradable inresponse to a second environmental condition, wherein the firstdegradable material differs from the second degradable material. In anaspect, the first degradable material and the second degradable materialare degradable in response to at least one of temperature, pH, chemicalreaction, electric field or electromagnetic energy.

In an aspect, the particles of the first set of particles 110 and of theat least one second set of particles 120 have a structure formed from amaterial capable of changing from a first phase or state to a secondphase or state in response to a change in environmental condition or astimulus. For example, the particles forming each of the two or moresets of particles can be formed from a material that changes from asolid or semi-solid state to a fluid state in response to a change inenvironmental condition or a stimulus. For example, the particlesforming each of the two or more sets of particles can be formed from amaterial that changes from a fluid state to a solid or semi-solid statein response to a change in environmental condition or a stimulus. Forexample, the particles forming each of the two or more sets of particlescan be formed from a material that changes phase or state in response toa change in temperature, a change in pH, a change in electric field,exposure to electromagnetic energy (e.g., light of a specificwavelength), a chemical (e.g., a chemical that cleaves a cross-linker ofa polymer), an enzyme, and the like.

In an aspect, the particles of the first set of particles 110 and of theat least one second set of particles 120 include a structure formed froma temperature-responsive degradable material. For example, the particlesforming each of the two or more sets of particles 105 can include astructure that at least one of degrades, melts, liquefies, disperses,dissolves, decomposes, disintegrates, breaks apart, deforms, or changesphase in response to increasing or decreasing temperature from a firsttemperature to a second temperature. In an aspect, the particles of thefirst set of particles 110 and of the at least one second set ofparticles 120 are degradable in response to increasing or decreasing atemperature from a first temperature to a second temperature by about 5°C. to about 100° C. In an aspect, the particles forming each of the twoor more sets of particles 105 are formed from a material or materialsthat degrades in response to increasing or decreasing a temperature froma first temperature to a second temperature by about 5° C. to about 100°C. For example, each particle of the first set of particles and the atleast one second set of particles is degradable in response to anincrease or a decrease in temperature of at least about 5° C., at leastabout 10° C., at least about 15° C., at least about 20° C., at leastabout 25° C., at least about 30° C., at least about 35° C., at leastabout 40° C., at least about 45° C., at least about 50° C., at leastabout 55° C., at least about 60° C., at least about 65° C., at leastabout 70° C., at least about 75° C., at least about 80° C., at leastabout 85° C., at least about 90° C., at least about 95° C., or at leastabout 100° C., or any other temperature differential that allows theparticles to degrade. As an example, the first set of particles and thesecond set of particles can be formed from heated agarose that forms asemi-solid gel upon cooling but can be re-liquefied in response towarming. Other non-limiting examples of temperature sensitive orresponsive polymers include poly(N-isopropylacrylamide),poly(N-vinylcaprolactam), poly(lactic acid), poly(N-ethylacrylamide),poly(N-cyclopropymethacrylamide), poly(N-methyl-N-ethylacrylamide),poly(N-acryloylpyrrolidine), poly(N-ethylmethacrylamide),poly(N-cyclopropylacrylamide), poly(N-isopropylmethacrylamide),poly(N,N-diethylacrylamide), poly(N-n-propylmethacrylamide),poly(N-methyl-N-isopropylacrylamide),poly(N-methyl-N-isopropylacrylamide), poly(N-n-propylacrylamide),poly(N-methyl-N-n-propylacrylamide), poly(N-acryloylpiperidine), and thelike.

In an aspect, the particles of the first set of particles 110 and of theat least one second set of particles 120 include a structure formed froma pH-responsive degradable material. For example, the particles formingeach of the two or more sets of particles 105 can include a structurethat at least one of degrades, melts, liquefies, disperses, dissolves,decomposes, disintegrates, breaks apart, deforms, or changes phase inresponse to increasing or decreasing the pH from a first pH to a secondpH. In an aspect, particles of the first set of particles and the atleast one second set of particles is degradable in response toincreasing or decreasing the pH of the environment by about 0.5 pH unitsto about 14 pH units. In an aspect, particles forming each of the two ormore sets of particles 105 are formed from a material or materials thatdegrades in response to increasing or decreasing the pH of theenvironment by about 0.5 pH units to about 14 pH units. For example, theparticles of the first set of particles and of the at least one secondset of particles are degradable in response to an increase or a decreasein pH units of at least about 0.5 pH units, of at least about 1 pHunits, of at least about 3 pH units, of at least about 4 pH units, of atleast about 5 pH units, of at least about 6 pH units, of at least about8 pH units, of at least about 10 pH units, of at least about 12 pHunits, or of at least about 14 pH units, or any other change in pH unitsthat allows the particles to degrade. Non-limiting examples of gels orpolymers that at least partially degrade in response to a change in pHinclude aminoalkyl methacrylate, poly(methacrylic acid-co-methylmethacrylate), hydroxypropyl-methylcellulose phthalate,hydroxypropyl-methylcellulose acetate succinate, poly(acrylate),poly(acetoacetoxyethyl methacrylate), poly[2-(diisopropylamino)ethylmethacrylate], poly(hexyl methacrylate), and poly[2-(dimethylamine)ethylmethacrylate).

In an aspect, the particles of the first set of particles 110 and of theat least one second set of particles 120 include a structure formed froma chemically-responsive degradable material. For example, the particlesforming each of the two or more sets of particles 105 can include astructure that at least one of degrades, degrades, melts, liquefies,disperses, dissolves, decomposes, disintegrates, breaks apart, deforms,or changes phase in response to a chemical reaction. In an aspect, thechemical reaction includes a change in pH or ionic strength. Forexample, the particles can be formed from a pH-responsive material,examples of which have been described above. In an aspect, the chemicalreaction includes cleaving a cross-linker. For example, the particlescan be formed from a material with cleavable cross-linking. For example,the particles can include disulfide cross-linking degradable in responseto reducing agents such as, for example, dithiothreitol. Otherdegradable links include anhydrides, imines, oximes, acetals,hydrazides, hydrazines, hydrazones. For example, the particles caninclude or be formed from degradable oligo/polymer segments such as, forexample, chitin, chitosan, a polysaccharide, a peptide or protein, orpolyesters. In an aspect, the particles are formed from a materialdegradable in response to an enzymatic reaction, e.g., in response to aprotease or peptidase, a chitinase, or an amylase.

In an aspect, the particles of the first set of particles 110 and of theat least one second set of particles 120 include a structure formed froman electric field-responsive degradable material. For example, theparticles forming each of the two or more sets of particles 105 caninclude a structure that at least one of degrades, degrades, melts,liquefies, disperses, dissolves, decomposes, disintegrates, breaksapart, deforms, or changes phase in response to application of anelectric field. For example, the particles can be formed from a materialthat changes form, e.g., shrinks or swells, in response to applicationof an electric field. Non-limiting examples of electro-responsivepolymers include poly(dimethylsiloxane), poly[2-(methacryloyloxy)ethylphosphorylcholine], and poly(ethylenediamine-co-1,10-bis(chloro-carbonyl)decane).

In an aspect, the particles of the first set of particles 110 and of theat least one second set of particles 120 include a structure formed froman electromagnetic energy-responsive degradable material. For example,the particles forming each of the two or more sets of particles 105 caninclude a structure that at least one of degrades, melts, liquefies,disperses, dissolves, decomposes, disintegrates, breaks apart, deforms,or changes phase in response to electromagnetic energy. For example, theparticles can be formed from a photo-responsive material that changesproperties when irradiated with ultraviolet or visible light of anappropriate wavelength. For example, the particles can be formed frompolymers including photoactive groups, such as, for example, azobenzene,spirobenzopyran, triphenylmethane or cinnamonyl groups. Non-limitingexamples of photo-responsive polymers includepoly(NN-dimethylacrylamide-co-4-phenyl-azophenyl acrylate) andpoly(N,N-dimethylacrylamide-co-4-phenyl-azophenyl acrylamide).

In an aspect, the particles of the first set of particles 110 and of theat least one second set of particles 120 are hydrophilic. In an aspect,the particles of the first set of particles 110 and of the at least onesecond set of particles 120 include a structure at least partiallyformed from a hydrophilic material. In an aspect, the entirety of eachparticle is formed from a hydrophilic degradable material. In an aspect,the outer surface of each particle is formed from a hydrophilicdegradable material. For example, the particles can be formed fromhydrophilic polymer chains, e.g., a hydrogel, non-limiting examples ofwhich have been described above herein. For example, the particles canbe formed from a neutral hydrophilic polymer, a non-limiting example ofwhich includes agarose. In an aspect, at least a portion of theparticles of the first set of particles 110 and of the at least onesecond set of particles 120 are distributable into an aqueous portion ofaqueous-in-oil droplets. For example at least a portion of the particlesin the two or more sets of particles partition into the aqueous portionof aqueous-in-oil droplets.

Returning to FIG. 1 , the first set of particles 110 includes a firstset of one or more target-specific reagents 114 selected to specificallyinteract with a first target and the at least one second set ofparticles 120 includes a second set of one or more target-specificreagents 124 selected to specifically interact with a second target. Forexample, the one or more target-specific reagents are configured to,designed to, or capable of interaction with a target. For example, theone or more target specific reagents can include a sequence (e.g., anuclei acid or amino acid sequence), a three dimensional structure, oran affinity that enables them to specifically interact with a target. Inan aspect, the one or more target-specific reagents react with a targetby binding to the target. For example, the one or more target-specificreagents can have a nucleic acid sequence or sequences selected to,capable of, or designed to bind or hybridize to a specific target, e.g.,a nuclei acid sequence. For example, the one or more target-specificreagents can include complimentary nuclei acid sequences that at leastpartially hybridize to a corresponding target nucleic acid sequence. Forexample, the first set of one or more target-specific reagents can beselected to specifically interact with (e.g., bind to) or hybridize to afirst nucleic acid sequence and the second set of one or moretarget-specific reagents can be selected to specifically interact with(e.g., bind to) or hybridize to a second nucleic acid sequence. Forexample, the one or more target-specific reagents can be configured,selected, or designed to bind to an epitope or similar structure on aspecific target. For example, the one or more target-specific reagentscan have an amino acid sequence or sequences and a correspondingconfiguration or three-dimensional structure capable of binding to anepitope or similar structure on a specific target. For example, thefirst set of one or more target-specific reagents can be configured,selected, or designed to specifically bind to a first antigen and thesecond set of one or more target-specific reagents can be configured,selected, or designed to specifically bind to a second antigen. Forexample, the one or more target-specific reagents can be configured,selected, or designed to bind into an active site of a specific target.For example, the one or more target-specific reagents can have achemical composition and/or physical structure that confers a bindingaffinity for a specific target. For example, the one or moretarget-specific reagents can be configured, selected, or designed tobind to a receptor, e.g., a receptor on the surface of a cell. Forexample, the one or more target-specific reagents can serve as acatalyst or substrate or primer for a reaction. For example, the one ormore target-specific reagents can include an agonist or antagonist of acellular or biochemical reaction. For example, the one or moretarget-specific reagents can include an inhibitor of bacterialproliferation and/or growth.

In an aspect, the one or more target-specific reagents in the first setof one or more target-specific reagents 114 and in the second set of oneor more target-specific reagents 124 are selected to interact with thesurface of a specific cell type or types. For example, the one or moretarget-specific reagents are configured, selected, or designed tointeract with (e.g., bind to) the surface of a specific cell type ortypes. For example, the one or more target-specific reagents areconfigured, selected, or designed to interact with (e.g., bind to) thesurface of a specific cell type or types. In an aspect, the one or moretarget-specific reagents are selected to interact with (e.g., bind to) aspecific biomolecule(s) associated with the surface of a microbe. Forexample, the target-specific reagents can be selected to specificallyinteract with biomolecules (e.g., proteins, lipids, carbohydrates,nucleic acids) on the surface of bacteria, a virus, a fungus, and/or aparasite. In an aspect, the one or more target-specific reagents areselected to specifically interact with biomolecules (e.g., proteins,lipids, carbohydrates, nucleic acids) associated with the surface ofmammalian cells. The mammalian cells can be derived from a body fluid,swab, or excised tissue and can include, but not limited to, red bloodcells, platelets, white blood cells, inflammatory cells, cancerouscells, normal tissue cells, tumor cells, and the like. In someembodiments, the multiplexed analysis includes one or more steps oflysing a cell or cells to allow interaction of the one or moretarget-specific reagents with biomolecules internal to the surface ofthe cell.

In an aspect, the one or more target-specific reagents in the first setof one or more target-specific reagents 114 and in the second set of oneor more target-specific reagents 124 include oligonucleotides,antibodies, aptamers, or combinations thereof. Other non-limitingexamples of target-specific reagents includes antibody fragments,peptides, peptide nucleic acids, proteins, viruses, bacteriophage,phospholipids, carbohydrates, enzymes, substrates, receptors, lectins,peptide aptamers, inorganic molecules, organic molecules, small moleculeagonists or antagonists, or combinations thereof.

In an aspect, the one or more target-specific reagents in the first setof one or more target-specific reagents 114 and in the second set of oneor more target-specific reagents 124 include one or moreoligonucleotides. The oligonucleotides can include DNA or RNA oligomers.For example, the one or more target-specific reagents can include one ormore oligonucleotides having a sequence compatible with or capable ofbinding to and/or detecting a specific nucleic acid sequence. In anaspect, the one or more target-specific oligonucleotides include one ormore oligonucleotide primers. The oligonucleotide primers can be RNA orDNA nucleotide sequences of 10 to 40 bases, wherein at least a portionof the nucleotide sequence is complimentary to a target nucleic acidsequence. In an aspect, the one or more target-specific reagents in thefirst set of target-specific reagents 114 and in the second set oftarget-specific reagents 124 comprise amplification primer sets. Forexample, the one or more target-specific oligonucleotides can includeone or more primer sets for priming amplification of a specific nucleicacid sequence in a sample. For example, the first set of one or moretarget-specific reagents can include a first amplification primer setconfigured, selected, or designed to prime amplification of a firstnucleic acid sequence and the second set of one or more target-specificreagents can include a second amplification primer set configured,selected, or designed to prime amplification of a second nucleic acidsequence.

In some embodiments, system 100 includes components capable of ordesigned for multiplexed detection of two or more nucleic acid sequencesin a test sample. In some embodiments, system 100 includes the two ormore sets of particles 105, wherein each particle 112 a-112f of thefirst set of particles 110 includes a first set of one or moretarget-specific reagents 114 that is a first amplification primer setselected to specifically interact with a first nucleic acid sequence andeach particle 122 a-122f of the at least one second set of particles 120includes a second set of one or more target-specific reagents 124 thatis a second amplification primer set selected to specifically interact asecond nucleic acid sequence. Each particle 112 a-112f of the first setof particles 110 further includes a first optically detectableidentifier 116 capable of emitting a first wavelength that is indicativeof the first amplification primer set and each particle 122 a-122f ofthe at least one second set of particles 120 further includes a secondoptically detectable identifier 126 capable of emitting a secondwavelength that is indicative of the second amplification primer set.System 100 can further include at least one optically detectablereporter probe 130 capable of constitutively emitting a third wavelengthin response to amplification of the first nucleic acid sequence in thetest sample and/or the second nucleic acid sequence in the test sample.

In an aspect, the one or more target-specific reagents in the first setof one or more target-specific reagents 114 and in the second set of oneor more target-specific reagents 124 comprise antibiotics. For example,the particles of the two or more sets of particles 105 can include oneor more antibiotics or similar agents having at least one ofbactericidal or bacteriostatic activity against bacteria or subsets ofbacteria. For example, each set of particles can include an antibioticor antibiotics with specificity for a particular bacteria or class ofbacteria for assessing the types of bacteria in a test sample.Alternatively, antibiotics in the two or more sets of particles can beused for multiplexed analysis of antibiotic resistance against one ormore subsets of bacteria. Non-limiting examples of antibiotics includeaminoglycosides, ansamycins, carbapenems, cephalosporins, glycopeptides,lincosamides, macrolides, beta-lactams, monobactams, tetracyclines,sulfonamides, quinolones, penicillins, nitrofurans, and oxazolidinones.

In some embodiments, system 100 includes components capable of ordesigned for multiplexed analysis of antibiotic resistance in abacterial sample. In an embodiment, system 100 includes two or more setsof particles 105, wherein each particle 112 a-112f of the first set ofparticles 110 includes a first set of target-specific reagents 114 thatincludes a first antibiotic having bactericidal or bacteriostaticactivity against a first subset of bacteria and each particle 122 a-122fof the at least one second set of particles 120 includes a second set oftarget-specific reagents 124 that includes a second antibiotic havingbactericidal or bacteriostatic activity against a second subset ofbacteria. Each particle 112 a-112f of the first set of particles 110further includes a first optically detectable identifier 116 capable ofemitting a first wavelength that is indicative of the first antibioticand each particle 122 a-112f of the at least one second set of particles120 further includes a second optically detectable identifier 126capable of emitting a second wavelength that is indicative of the secondantibiotic. System 100 can further include at least one opticallydetectable reporter probe 130 capable of constitutively emitting a thirdwavelength in response to viability in the bacterial sample.

In an aspect, the one or more target-specific reagents in the first setof one or more target-specific reagents 114 and in the second set of oneor more target-specific reagents 124 comprise one or more agonists orantagonists. For example, the one or more target-specific reagents caninclude one or more agonists or antagonists of a cellular function(e.g., proliferation, inflammatory response, enzymatic activity, ionflux). In some embodiments, each set of the two or more sets ofparticles includes one or more target-specific reagents that interactwith the same target, e.g., a receptor or enzyme, but with variedaffinity and activity, allowing for multiplexed analysis of the variedtarget-specific reagents against a particular biochemical reaction. Forexample, each set of particles can include a specific agonist orantagonist for use in multiplexed analysis of an array of agonists orantagonists against a particular biochemical reaction. In an aspect, thetwo or more sets of particles are designed forhigh-throughput/multiplexed analysis of antagonists and/or agonistsagainst one or more targets.

In an aspect, the one or more target-specific reagents in the first setof one or more target-specific reagents 114 and in the second set of oneor more target-specific reagents 124 comprise antibodies. An antibodythat “specifically binds to” or is “specific for” a particular target orepitope on a particular target is one that binds to a particular targetor epitope on a particular target without substantially binding to anyother target or epitope. The term “antibody” is used here in itsbroadest sense and includes, but is not limited to, polyclonalantibodies, single monoclonal antibodies, antibody compositions withpolyepitopic specificity, humanized antibodies, bispecific antibodies,heteroconjugate antibodies, single chain antibodies, and fragments ofantibodies, among others. Fragments of antibodies comprising a portionof an intact antibody can include, but are not limited to, the antigenbinding and/or variable region of the intact antibody. Non-limitingexamples of antibody fragments include Fab, Fab′, Fab₂, F(ab′)₂, Fv,single-chain variable fragments (scFvs), diabody fragments (dimers ofscFvs fragments), minibody fragments, (dimers of scFvs-C_(H)3 withlinker amino acid), or the like. Methods for generating antibodies andfragments thereof are well known to the skilled artisan. Antibodyfragments can be produced by modification of whole antibodies orsynthesized do novo using recombinant DNA technologies.

In an aspect, the one or more target-specific reagent in the first setof one or more target-specific reagent 114 and in the second set of oneor more target-specific reagents 124 comprises enzymes or enzymesubstrates. Target-specific reagents that include an enzyme or enzymescan be configured, selected, or designed to interact with a substrate orsubstrates in the test sample. Target-specific reagents that include anenzyme or enzymes can be configured, selected, or designed to interactwith agonists or antagonists in the test sample. For example, the systemincluding the two or more sets of particles can be used for drugscreening, e.g., screening for agonists or antagonists that modify anactivity of an enzyme or enzymes. Target-specific reagents that includean enzyme substrate or substrates can be configured, selected, ordesigned to interact with enzymes in the test sample.

In an aspect, the one or more target-specific reagents in the first setof one or more target-specific reagents 114 and in the second set of oneor more target-specific reagents 124 comprise aptamers. The aptamers canbe oligonucleotide RNA- or DNA-based aptamers selected or designed witha sequence capable of recognizing and binding to one or more targets inthe test sample. In an aspect, the one or more target-specific reagentsin the first set of one or more target-specific reagents 114 and in thesecond set of one or more target-specific reagents 124 comprisepeptide-based aptamers, an artificial protein in which inserted peptidesare expressed as part of the primary sequence of a structurally stableprotein and having binding affinities comparable to antibodies. See,e.g., Crawford, et al., Brief. Funct. Genomic Proteomic 2:72-79, 2003,which is incorporated herein by reference. In an aspect, the one or moretarget-specific reagents in the first set of one or more target-specificreagents 114 and in the second set of one or more target-specificreagents 124 include novel peptides generated by a combinatorialapproach and selected or designed to specifically recognize and bind atarget.

In an aspect, the one or more target-specific reagents in the first setof one or more target-specific reagents 114 and in the second set of oneor more target-specific reagents 124 include a ligand that specificallyrecognizes one or more microbes, e.g., bacteria. In an aspect, thetarget-specific reagent includes all or part of a pattern recognitionreceptor that recognizes microbe-specific molecules (e.g., bacterialcarbohydrates, bacterial or viral DNA or RNA, bacterial peptides,peptidoglycans, lipoteichoic acids, N-formylmethionine, lipoproteins,and fungal glucans). Non-limiting examples of pattern recognitionreceptors with microbe-binding properties include toll-like receptors,C-type lectin receptors, NOD-like receptors, RIG-I-like receptors, RNAhelicases, complement receptors, collectins, ficolins, pentraxins,C-reactive proteins, lipid transferases, and the like.

In an aspect, the one or more target-specific reagents in the first setof one or more target-specific reagents 114 and in the second set of oneor more target-specific reagents 124 include a lectin. Lectins includecarbohydrate-binding proteins that bind cell surface glycoproteinsand/or glycolipids and can be derived from plant, animal, bacterial,fungal, or viral sources.

In an aspect, each of the first set of one or more target-specificreagents 114 and of the second set of one or more target-specificreagents 124 includes a set of binding elements modified for proximitysignaling. For example, a set of target-specific reagents can include afirst binding element configured, selected, or designed to bind a firsttarget and a second binding element configured, selected, or designed tobind a second target, wherein the first target and the second target areproximal to one another and binding of the first binding element and thesecond binding element to their respective targets directly orindirectly results in an optically detectable signal. In someembodiments, the first target and the second target are on the samemolecule, e.g., different epitopes on the same antigen. In someembodiments, the first target and the second target are on differentmolecules, e.g., epitopes on different antigens. Non-limiting examplesof binding elements include oligonucleotides, antibodies, aptamers,peptides, proteins, ligands, and lectins. The binding elements can befurther modified to provide proximity signaling. For example, thebinding elements can be modified with oligonucleotides capable ofpriming amplification when in close proximity For example, the bindingelements can be modified with a donor-acceptor pair selected or designedto generate or shift a fluorescent signal when in close proximity

In an aspect, each of the first set of one or more target-specificreagents 114 and of the second set of one or more target-specificreagents 124 includes a set of antibodies for antibody-based proximitysignaling. For example, each set of one or more target specific reagentscan include a set of antibodies which bind different epitopes on atarget, e.g., a target protein. For example, each set of one or moretarget-specific reagents can include a set of antibodies which binddifferent targets (e.g., different receptors) on a cell surface (e.g.,on the surface of an immune cell), wherein the targets are proximal toone another. In some embodiments, a first antibody of the set ofantibodies is modified with a first molecule and a second antibody ofthe set of antibodies is modified with a second molecule, wherein whenthe first antibody and the second antibody bind to their respectiveproximal targets, the first molecule and the second molecule are able tointeract. In some embodiments, the interaction of the first molecule andthe second molecule directly or indirectly results in an opticallydetectable signal, e.g., fluorescence.

In some embodiments, system 100 includes components capable of ordesigned for multiplexed analysis of two or more antigens in a testsample. In an aspect, system 100 includes two or more sets of particles105, wherein each particle 112 a-112f of the first set of particles 110includes a first set of target-specific reagents 114 that includes afirst antibody set including two or more antibodies specific forproximal targets on a first antigen, the two or more antibodies of thefirst antibody set including modifications capable of interacting in anantibody-based proximity assay and each particle 122 a-122f of the atleast one second set of particles 120 includes a second set oftarget-specific reagents 124 that includes a second antibody setincluding two or more antibodies specific for proximal targets on asecond antigen, the two or more antibodies of the second antibody setincluding modifications capable of interacting in an antibody-basedproximity assay. Each particle 112 a-112f of the first set of particles110 further includes a first optically detectable identifier 116 capableof emitting a first wavelength that is indicative of the first antibodyset and each particle 122 a-112f of the at least one second set ofparticles 120 further includes a second optically detectable identifier126 capable of emitting a second wavelength that is indicative of thesecond antibody set. System 100 can further include at least oneoptically detectable reporter probe 130 that is capable ofconstitutively emitting a third wavelength in response to the two ormore antibodies of the first antibody set binding to their proximaltargets on the first antigen and/or the two or more antibodies of thesecond antibody set binding to their proximal targets on the secondantigen.

In an aspect, each of the first set of one or more target-specificreagents 114 and of the second set of one or more target-specificreagents 124 includes a set of antibodies modified witholigonucleotides. In some embodiments, a first antibody of the set ofantibodies is modified with a first oligonucleotide and a secondantibody of the set of antibodies is modified with a secondoligonucleotide, wherein when the first antibody and the second antibodybind to their respective proximal targets, the first oligonucleotide andthe second oligonucleotide are able to interact. For example, the firstmolecule and the second molecule associated with the antibodies caninclude oligonucleotides capable of at least partially hybridizing withone another when in proximity and form a template for amplification. Forexample, the first molecule and the second molecule associated with theantibodies can include oligonucleotides capable of being ligated to oneanother when in proximity and form a template for amplification.

In an aspect, each of the first set of one or more target-specificreagents 114 and of the second set of one or more target-specificreagents 124 includes a set of antibodies modified with areporter-quencher pair. In some embodiments, a first antibody of the setof antibodies is modified with a reporter and a second antibody of theset of antibodies is modified with a quencher, wherein when the firstantibody and the second antibody bind to their respective proximaltargets, the reporter and the quencher interact. For example, a firstantibody can include a fluorescent reporter and the second antibody caninclude a quencher and when the two antibodies bind to their respectiveproximal targets, the fluorescence associated with the fluorescentreporter is quenched. Non-limiting examples of reporter-quencher pairsare described below.

In an aspect, each of the first set of one or more target-specificreagents 114 and of the second set of one or more target-specificreagents 124 includes a set of antibodies modified with a donor-acceptorpair. In some embodiments, a first antibody of the set of antibodies ismodified with a donor and a second antibody of the set of antibodies ismodified with an acceptor, wherein when the first antibody and thesecond antibody bind to their respective proximal target, the donor andacceptor interact. For example, a first antibody can include a donormolecule that fluoresces at a first wavelength and the second antibodycan include an acceptor that fluoresces at a second wavelength inresponse to the first wavelength emitted by the donor.

In an aspect, the one or more target-specific reagents in the first setof one or more target-specific reagents 114 and in the second set of oneor more target-specific reagents 124 comprise bacteriophage. In anaspect, the target-specific reagent comprises a bacteriophage with abroad spectrum of bacterial infection. In an aspect, the target-specificreagent comprises a bacteriophage with a narrow spectrum of bacterialinfection. Additional information regarding the breadth of bacteriophagecan be found on various database websites, non-limiting examples ofwhich include the Actinobacteriophage Database (Russell & Hatfull (2017)Bioinformatics 33:784-786) and the MVP database (Gao, et al. (2018)Nucleic Acids Res. 46:D700-D707), the references to which areincorporated herein.

System 100 further includes a first optically detectable identifier 116associated with each of the first set of particles 110 and a secondoptically detectable identifier 126 associated with each of the at leastone second set of particles 120. Each of the optically detectableidentifiers is capable of emitting a wavelength (e.g., fluorescence of aspecific wavelength and color) and is indicative of the set of one ormore target-specific reagents associated with a given set of particles.Each of the optically detectable identifiers is further indicative ofthe specific target to which the set of one or more target-specificreagents is capable of reacting with. For example, a first opticallydetectable identifier capable of emitting a red wavelength of light maybe indicative of a first set of one or more target-specific reagents anda first target in a test sample, while a second optically detectableidentifier capable of emitting a green wavelength of light may beindicative of a second set of one or more target-specific reagents and asecond target in the test sample. The optically detectable identifieracts as an optical indicator of which target-specific reagents ispresent in a given particle type. The optically detectable identifier isan optical bar code for a given set of one or more target-specificreagents specific for a given target. The optically detectableidentifier can be at least one of a tag, a code, a label, an ID, or amarker indicative of the one or more target-specific reagents in a givenparticle type.

System 100 further includes at least one optically detectable reporterprobe 130 capable of constitutively emitting a third wavelength. In anaspect, the first wavelength emitted by the first optically detectableidentifier 116 is a first detectable color, the second wavelengthemitted by the second optically detectable identifier 126 is a seconddetectable color, and the third wavelength constitutively emitted by theat least one optically detectable reporter probe 130 is a thirddetectable color. In an aspect, the first wavelength, the secondwavelength, and the third wavelength are optically discernable from oneanother. In an aspect, the first wavelength, the second wavelength, andthe third wavelength are optically distinguishable, distinct, ordifferent from one another. As a non-limiting example, the firstoptically detectable identifier can emit a red color, the secondoptically detectable identifier can emit a green color, and the at leastone optically detectable reporter probe can constitutively emit a bluecolor.

System 100 includes two or more sets of particles 105 includingoptically detectable identifiers capable of emitting a wavelength and atleast one optically detectable reporter probe capable of constitutivelyemitting a wavelength. In an aspect, emitting a wavelength includesemitting a wavelength in response to temperature (incandescence),chemical reactions (chemiluminescence), biochemical reactions(bioluminescence), electrochemical reactions (electrochemiluminescence)or in response to absorbing light of other frequencies, e.g.,fluorescence, phosphorescence, and Raman emission. In an aspect,emitting a wavelength includes a color of light leaving a surface, i.e.,a wavelength or wavelengths reflected from a surface that is perceivedas color of said surface.

In an aspect, the optically detectable identifiers 116 and 126 and theat least one optically detectable reporter probe 130 are capable ofemitting a wavelength or wavelength band of electromagnetic radiation.In an aspect, the first wavelength, the second wavelength, or the thirdwavelength is an ultraviolet wavelength or wavelength band ofelectromagnetic energy. For example, the optically detectableidentifiers and/or the at least one optically detectable reporter probecan be capable of emitting a wavelength or wavelength band in the rangeof about 100 nm to about 400 nm. In an aspect, at least one of the firstwavelength, the second wavelength, or the third wavelength is a visiblewavelength or wavelength band of electromagnetic energy. For example,the optically detectable identifiers and/or the at least one opticallydetectable reporter probe can be capable of emitting a wavelength orwavelength band in the range of about 390 nm to about 750 nm. In anaspect, the first wavelength, the second wavelength, or the thirdwavelength is a near infrared wavelength or wavelength band ofelectromagnetic energy. For example, the optically detectableidentifiers and/or the at least one optically detectable reporter probecan be capable of emitting a wavelength or a wavelength band in therange of about 700 nm to about 2,500 nm). In an aspect, the emittedwavelength or wavelength band of electromagnetic radiation emitted fromthe optically detectable identifiers and/or the at least one opticallydetectable reporter probe includes radio waves, microwaves, X-rays, andgamma rays.

In an aspect, the first optically detectable identifier 116 is a firstcolored dye or pigment and the second optically detectable identifier126 is a second colored dye or pigment. Non-limiting examples of dyesinclude alcian yellow, alizarin, alizarin yellow, Bismarck brown,brilliant cresyl blue, congo red, crystal violet, fuchsin acid, gentianviolet, janus green, lassamine fast yellow, marius yellow, meldola blue,metanil yellow, methyl orange, methyl red, naphthol green, orange G,purpurin, rose bengal, titan yellow, Victoria blue, alizarine cyaninegreen, alizarine brilliant blue, and the like. Non-limiting examples ofpigments include metal-based pigment, an inorganic pigment, an organicpigment, or a biological pigment. In some embodiments, the pigmentitself is insoluble but dispersible as fine particles in an aqueousmedium. In an aspect, the pigment includes binders to facilitatedispersion.

In an aspect, the first optically detectable identifier 116 is a firstcolored particle and the second optically detectable identifier 126 is asecond colored particle. For example, the optically detectableidentifiers can include color-rich dyed particles (from, e.g.,ThermoFisher Scientific (Waltham, Mass.)).

In an aspect, the first optically detectable identifier 116 includes afirst fluorophore capable of emitting fluorescence at the firstwavelength and the second optically detectable identifier 126 includes asecond fluorophore capable of emitting fluorescence at the secondwavelength. In an aspect, the optically detectable identifiers includefluorescent dyes or fluorophores such as, for example, fluorescein(FITC), indocyanine green (ICG) and rhodamine B. Examples of otherfluorescent dyes or fluorophores include but are not limited to a numberof red and near infrared emitting fluorophores (600-1200 nm) includingcyanine dyes such as Cy5, Cy5.5, and Cy7 (Amersham Biosciences,Piscataway, N.J., USA) and/or a variety of Alexa Fluor dyes such asAlexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660,Alexa Fluor 680, Alexa Fluor 700 and Alexa Fluor 750 (MolecularProbes-Invitrogen, Carlsbad, Calif., USA; see, e.g., U.S. Pat. App. No.2005/0171434 A1). Additional fluorophores include IRDye800, IRDye700,and IRDye680 (LI-COR, Lincoln, Nebr., USA), NIR-1 and 1C5-OSu (Dejindo,Kumamotot, Japan), LaJolla Blue (Diatron, Miami, Fla., USA), FAR-Blue,FAR-Green One, and FAR-Green Two (Innosense, Giacosa, Italy), ADS 790-NSand ADS 821-NS (American Dye Source, Montreal, CA), NIAD-4 (ICxTechnologies, Arlington, Va.). Other fluorescing agents includeBODIPY-FL, europium, green, yellow and red fluorescent proteins.

In an aspect, the first optically detectable identifier 116 is a firstfluorescing microsphere capable of emitting fluorescence at the firstwavelength and the second optically detectable identifier 126 is asecond fluorescing microsphere capable of emitting fluorescence at thesecond wavelength. For example, the optically detectable identifiers caninclude FluoSpheres® fluorescing at blue, blue-green, yellow-green, Nilered, orange, red-orange, red, crimson, dark red, or near infrared (from,Thermo Fisher Scientific, Waltham, Mass.). Other examples includefluorescing microspheres from Ocean NanoTech (San Diego, Calif., USA).

In an aspect, the first optically detectable identifier 116 is a firstquantum dot capable of emitting at the first wavelength and the secondoptically detectable identifier 126 is a second quantum dot of emittingfluorescence at the second wavelength. For example, the first opticallydetectable identifier can include a first quantum dot type capable ofemitting fluorescence at the first wavelength and the second opticallydetectable identifier can include a second quantum dot type capable ofemitting fluorescence at the second wavelength. For example, theoptically detectable identifiers can include very small (nanoscale)semiconductor nanocrystal particles or fluorescing quantum dots. In anaspect, the quantum dots can be formed from one or more of lead sulfide(PbS), lead selenide (PbSe), cadmium selenide (CdSe), cadmium sulfide(CdS), cadmium telluride (CdTe), indium arsenide (CdAs), indiumphosphide (InP), copper indium sulfide (CuInS), silver sulfide (Ag2S),and zinc sulfide (ZnS). For example, the optically detectableidentifiers can include CdS/ZnS (emitting at 403-450 nm), CdSe/ZnS(emitting at 520-630 nm), CdSSe/ZnS (emitting at 450-665 nm), andInP/ZnS and PbS/Ag2S (emitting at 780-1600 nm). In an aspect, thequantum dots are coated or otherwise modified to improve aqueoussolubility. For example, the quantum dots can be modified withpolyethylene glycol (PEG), glutathione, dihydrolipoic acid, cysteine, or3-mercaptopropionic acid to improve solubility. In an aspect, thequantum dots are associated with a micro- or nano-particle.

System 100 further includes at least one optically detectable reporterprobe 130 capable of constitutively emitting a third wavelength inresponse to a reaction of the first set of one or more target-specificreagents 114 with the first target in a test sample and/or a reaction ofthe second set of one or more target-specific reagents 124 with thesecond target in the test sample. In an aspect, the at least oneoptically detectable reporter probe is capable of constitutivelyemitting a third wavelength in response to a reaction of the first setof one or more target-specific reagents with the first target or inresponse to a reaction of the second set of one or more target-specificreagents with the second target; or the at least one opticallydetectable reporter probe is capable of constitutively emitting a thirdwavelength in response to a reaction of the first set of one or moretarget-specific reagents with the first target and in response to areaction of the second set of one or more target-specific reagents withthe second target. The optically detectable reporter probe is designedto constitutively emit the third wavelength as long as at least onetarget is present to interact with target-specific reagents, but willalso emit the third wavelength if at least one second target is presentto interact with its respective target-specific reagents. The opticallydetectable reporter probe constitutively emits a wavelength (e.g., awavelength or wavelength band of ultraviolet, visible light, or nearinfrared electromagnetic energy) in response to interaction of the oneor more target-specific reagents with their intended target. In anaspect, the at least one optically detectable reporter probe is ageneric or universal probe of a particular reaction type. For example,the at least one optically detectable reporter probe can be a general,generic, or universal probe of amplification reactions, bindingreactions, enzymatic reactions, proliferation or cell viabilityreactions, and the like. For example, the at least one opticallydetectable reporter probe can include an intercalating reagent thatemits a wavelength, (e.g., fluoresces) in response to the formation andincreased concentration of double stranded DNA as a result ofamplification of any DNA or RNA in the test sample. For example, the atleast one optically detectable reporter probe can include a generic oruniversal marker of cell viability, e.g., a vital dye.

In some embodiments, the at least one optically detectable reporterprobe 130 is a separate component of system 100 relative to the firstset of particles 110 and the second set of particles 120. In someembodiments, the at least one optically detectable reporter probe 130 isassociated with each particle of the first set of particles 110 and witheach particle of the at least one second set of particles. For example,the at least one optically detectable reporter probe can be incorporatedinto the first and the at least one second set of particles duringformation of the particles. For example, the at least one opticallydetectable reporter probe can be mixed with the set of one or moretarget-specific reagents and the optically detectable identifier priorto forming the particles.

In an aspect, the at least one optically detectable reporter probe isspecific for a given set of one or more target-specific reagents and/orthe specific target. For example, the at least one optically detectablereporter probe can include a TaqMan-like probe that binds to a specificnucleic acid sequence. In an aspect, the at least one opticallydetectable reporter probe 130 includes a first optically detectablereporter probe specific for the first target and a second opticallydetectable reporter probe specific for the second target. For example, afirst optically detectable reporter probe can include a first TaqManprobe specific for a first nucleic acid sequence and a second opticallydetectable reporter probe can include a second TaqMan probe specific fora second nucleic acid sequence.

In an aspect, the at least one optically detectable reporter probe 130comprises a DNA intercalating agent capable of constitutively emittingthe third wavelength. For example, the at least one optically detectablereporter probe can include a DNA intercalating agent that is afluorescing DNA-binding agent. The DNA intercalating agent can includeany of a number of agents that preferentially bind to or intercalateinto double stranded DNA and can be used as an indicator ofamplification of a nucleic acid sequence. In some embodiments, the DNAintercalating agent can be used for quantification of amplification of anucleic acid sequence. Non-limiting examples of fluorescing DNAintercalating agents include ethidium bromide, propidium iodide, DAPI,SYTO-9, SYTO-13, SYTO-82, SYBR Green 1, SYBR Gold, EvaGreen, andacridine orange.

In an aspect, the at least one optically detectable reporter probe 130comprises a donor-acceptor pair capable of constitutively emitting thethird wavelength. For example, the optically detectable reporter probecan include a donor-acceptor pair which constitutively emits awavelength as long as the pair are in close proximity to one another bythe process of fluorescence resonance energy transfer (FRET). FRET is adistance-dependent interaction between the electronic excited states oftwo dye molecules in which excitation is transferred from a donormolecule to an acceptor molecule without emission of a photon. In anaspect, interaction of a donor molecule with an acceptor molecule canlead to a shift in the emission wavelength associated with excitation ofthe acceptor molecule. In an aspect, interaction of a donor moleculewith an acceptor molecule can lead to quenching of the donor emission.In some embodiments, the donor-acceptor pair are located on the samemolecule and changes in the configuration or conformation of themolecule upon interacting with a target changes the distance between thedonor and acceptor. In some embodiments, the donor and the acceptor arelocated on separate molecules. For example, the donor can be located ona first molecule (e.g., an antibody or aptamer) and the acceptormolecule can be located on a second molecule (a second antibody oraptamer) and when the first and second molecules are in proximity to oneanother, the emitted signal (e.g., fluorescence) from the donor-acceptorpair is altered (e.g., shifted or quenched).

A variety of donor and acceptor fluorophore pairs can be considered forFRET including, but not limited to, fluorescein andtetramethylrhodamine; IAEDANS and fluorescein; fluorescein andfluorescein; and BODIPY FL and BODIPY FL. A number of Alexa Fluor (AF)fluorophores (Molecular Probes-Invitrogen, Carlsbad, Calif., USA) can bepaired with other AF fluorophores for use in FRET. Some examplesinclude, but are not limited, to AF 350 with AF 488; AF 488 with AF 546,AF 555, AF 568, or AF 647; AF 546 with AF 568, AF 594, or AF 647; AF 555with AF594 or AF647; AF 568 with AF6456; and AF594 with AF 647.

Other non-limiting examples of fluorophores for FRET-based signalinginclude cyanine dyes Cy3, Cy5, Cy5.5 and Cy7, which emit in the red andfar red wavelength range (>550 nm). For example, Cy3, which emitsmaximally at 570 nm and Cy5, which emits at 670 nm, can be used as adonor-acceptor pair. When Cy3 and Cy5 are not proximal to one another,excitation at 540 nm results only in the emission from of light from Cy3at 590 nm. In contrast, when Cy3 and Cy5 are brought into proximity by aconformation change, e.g., by binding of a microbe to a specificmicrobe-binding element, excitation at 540 nm results in an emission at680 nm.

In some embodiments, the donor-acceptor pair includes afluorophore-quencher pair. In an aspect, the at least one opticallydetectable reporter probe 130 comprises a fluorophore-quencher paircapable of constitutively emitting the third wavelength. For example,the optically detectable reporter probe can include afluorophore-quencher pair in which the quencher molecule quenchesfluorescence emitted by the fluorophore as long as the pair are in closeproximity to one another by the process FRET. Upon increasing thedistance between the pair, the fluorescence from the fluorophore becomesdetectable. In some embodiments, the fluorophore-quencher pair arelocated on the same molecule and changes in the configuration orconformation of the molecule upon interacting with a target changes thedistance between the fluorophore and quencher. In some embodiments, thefluorophore and the quencher are located on separated molecules. Forexample, the fluorophore can be located on first molecule (e.g., anantibody or aptamer) and the quencher can be located on a secondmolecule (a second antibody or aptamer) and when the first and secondmolecules are in proximity to one another, the fluorophore is quenched.If the binding targets of the first molecule and the second molecule arein close proximity, binding of these reagents to their respectivetargets will bring the fluorophore and quencher into proximity of oneanother, leading to a decrease in detected fluorescence.

In some embodiments, the at least one optically detectable reporterprobe includes a fluorophore-quencher pair that constitutively emits awavelength in response to a cleavage reaction. In an aspect, the atleast one optically detectable reporter probe includes at least oneoligonucleotide probe with a cleavable fluorophore-quencher pair. Forexample, the at least one optically detectable reporter probe caninclude at least one TaqManTm probe designed to anneal with a portion ofa nucleic acid sequence to be amplified by a specific primer set and torelease the fluorophore in response to exonuclease activity of a DNApolymerase (from Thermo Fisher Scientific, Waltham, Mass.). Othercommercially available probe systems that constitutively emit awavelength or wavelength band in response to interacting with a nucleicacid sequence during an amplification reaction include, but are notlimited to, LightCycler® Probes and Scorpions® Probes (fromSigma-Aldrich, Corp. St. Louis, Mo.) and MGB Eclipse® Probes (fromIntegrated DNA Technologies, Skokie, Ill.).

In some embodiments, the at least one optically detectable reporterprobe includes a fluorophore-quencher pair that constitutively emits awavelength in response to a structural confirmation change in the probe.In an aspect, the at least one optically detectable reporter probeincludes a molecular beacon. For example, the at least one opticallydetectable reporter probe can include at least one oligonucleotidesequence with a stem-loop structure in the absence of binding, e.g.,hybridizing, to a target and having a 5′ fluorophore and a 3′ quencher.Upon binding to a target sequence, the stem-loop structure is disrupted,the fluorophore and the quencher become spatially separated, resultingin emission of detectable fluorescence from the fluorophore.

In an aspect, the at least one optically detectable reporter probeincludes an antibody, aptamer, or other binding entity including afluorophore-quencher pair. For example, the at least one opticallydetectable reporter probe can include an antibody with a fluorophore anda quencher in close proximity to one another which separate in responseto either degradation of the antibody upon cellular internalization or aconformational change in the antibody when it binds to its specifictarget.

A variety of fluorophore-quencher pairs can be considered forconstitutive emission of a wavelength through FRET. Non-limitingexamples include Cal Fluor Cold 540 or Cal Fluor Orange 560 paired withBHQ-1; 6-FAM, JOE, TET, or HEX paired with BHQ-1, DABCYL, or TAMRA;Cyanine 3, ROX, or TxRd paired with BHQ-2 or DABCYL; or Cyanine 5 or 5.5paired with BHQ-3 or DABCYL. Other non-limiting examples of fluorophoreand quencher pairs include fluorescein with DABCYL; EDANS with DABCYL;or fluorescein with QSY 7 and QSY 9. In general, QSY 7 and QSY 9 dyesefficiently quench the fluorescence emission of donor dyes includingblue-fluorescent coumarins, green- or orange-fluorescent dyes, andconjugates of the Texas Red and Alexa Fluor 594 dyes. QSY 21 dyeefficiently quenches all red-fluorescent dyes (from, e.g., MolecularProbes, Carlsbad, Calif., USA).

In an aspect, the at least one optically detectable reporter probe is anRNA or DNA oligonucleotide-based aptamer that includes afluorophore-quencher pair. See, e.g., Cao et al. (2005) CurrentProteomics 2:31-40 and U.S. Patent Application 2009/0186342, which areincorporated herein by reference. For example, the aptamer including afluorophore-quencher pair can have a sequence designed to undergo aconformational change upon binding a target, causing the distancebetween the fluorophore and the quencher to shift, leading to a changein measurable fluorescence.

In an aspect, the at least one optically detectable reporter probe 130comprises a probe of cell viability capable of constitutively emittingthe third wavelength in response to cell viability. For example, theoptically detectable reporter probe can be a generic or universal probeof cell viability. For example, the optically detectable reporter probecan be a live/dead cell indicator. For example, the optically detectablereporter probe can be a viability/cytotoxicity indicator. For example,the optically detectable reporter probe can constitutively reportwhether a cell population is alive or dead.

In an aspect, the at least one optically detectable reporter probe 130is a probe of cell viability that includes an indicator of cell membraneintegrity. The loss of cell membrane integrity is correlated with celldeath. For example, the optically detectable reporter probe can includea vital dye, e.g., trypan blue, which is excluded from cells until theintegrity of the cell membrane is compromised. Other non-limitingexamples of vital dyes includes eosin, propidium iodide, erythrosine,aminoactinomycin D, indocyanine green, Brilliant blue, and Janus greenB. For example, the optically detectable reporter probe can includered-fluorescent ethidium homodimer-1.

In an aspect, the at least one optically detectable reporter probe 130is a probe of cell viability that includes a substrate for an enzymaticactivity associated with dead or dying cells. For example, the opticallydetectable reporter probe can include a substrate of lactosedehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, or adenylatekinase that releases a fluorescent, chemiluminescent, or bioluminescentproduct in response to enzymatic activity. In an aspect, the at leastone optically detectable reporter probe 130 can include a generic oruniversal reporter of intracellular activity. For example, the at leastone optically detectable reporter probe can include green-fluorescentcalcein-AM to indicate intracellular esterase activity.

In some embodiments, the at least one optically detectable reporterprobe 130 is a fluorophore-labeled bacteriophage. For example, the DNAof a bacteriophage can be labeled with a fluorescent dye or fluorophore(e.g., YOYO-1, DAPI, or SYBR Green) and used to bind to the surface ofbacteria (prior to actual infection). For other non-limiting examples ofreporting via labeling of phage DNA, see, e.g., Smartt & Ripp (2011)Anal. Bioanal. Chem. 400:991-1007, which is incorporated herein byreference.

In an aspect, the at least one optically detectable reporter probe 130comprises a bacteriophage with a reporter gene capable of constitutivelyemitting the third wavelength. For example, a reporter gene or genes canbe incorporated into the phage genome and upon infection of bacteria,the reporter gene(s) carried by the bacteriophage is expressed. As such,bacteriophage including a reporter gene can be used as an indicator ofbacterial viability. In an aspect, the bacteriophage includes a reportergene that generates a colorimetric, fluorescent, chemiluminescent, orbioluminescent signal in response to infection and propagation of viablebacterial cells. In some instances, the response is spontaneous (e.g.,autofluorescence associated with green fluorescent protein). In someinstances, the response requires a substrate or cofactor, eitherendogenous or exogenous to the bacteria (e.g., when the reporter gene isan enzyme). Non-limiting examples of reporter genes include thoseencoding green, yellow or red fluorescent protein (GFP, YFP, or RFP,respectively), bacterial luciferase, firefly luciferase,beta-galactosidase (lacZ), chloramphenyl acetyltransferase (CAT),beta-glucuronidase (GUS). For other non-limiting examples of reportingvia reporter genes, see, e.g., Smartt & Ripp (2011) Anal. Bioanal. Chem.400:991-1007, which is incorporated herein by reference.

In an aspect, the at least one optically detectable reporter probe 130comprises a substrate capable of constitutively emitting the thirdwavelength in response to interaction with an enzyme. In an aspect, thesubstrate is at least one of a chemical substrate, a lipid-basedsubstrate, a peptide-based substrate, or a protein-based substrate. Inan aspect, the interaction of the substrate with a target enzymeproduces a colorimetric or fluorogenic product. In an aspect, theinteraction of the substrate with a target enzyme is detected bychemiluminescence. In an aspect, the at least one optically detectablereporter probe includes a substrate for at least one ofbeta-glucuronidase, beta-glucosidase, beta-galactosidase,beta-lactamase, beta-glucuronidase, alkaline phosphatase, luciferase,cytochrome P450, deubiquitinating enzyme, kinase, phosphatase, lipase,phospholipase, protease, or peptidase. In an aspect, the at least oneoptically detectable reporter probe includes a substrate that interactswith a component of a cell membrane (e.g., lipids, proteins and proteinreceptors, and carbohydrates). A number of substrates whichconstitutively emit a wavelength in response to interaction with anenzymatic target are available from commercial sources (from, e.g.,Thermo Fisher Scientific, Waltham, Mass.).

In an aspect, the at least one optically detectable reporter probe 130includes a substrate for beta-glucuronidase. For example, the substratefor beta-glucuronidase can include a substrate that generates acolorimetric precipitate in response to the interaction. Non-limitingexamples include 5-bromo-4-chloro-3-indolyl-betα-D-glucuronide (X-GlcA),para-nitrophenyl-β-D glucuronide (pNPG), andphenolphthalein-β-D-glucuronide (PHTG). For example, the substrate forbeta-glucuronidase can include a substrate that generates a fluorogenicproduct in response to the interaction. A non-limiting example includes4-methylumbelliferyl-β-D-glucuronide (MUG).

In an aspect, the at least one optically detectable reporter probe 130includes a substrate for beta-galactosidase. For example, the substratefor beta-galactosidase can include a substrate that generates acolorimetric precipitate in response to the interaction, non-limitingexamples of which include5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal), halogenatedindolyl-β-galactoside, or ortho-nitrophenyl-β-D-galactopyranoside(ONPG). For example, the substrate of beta-galactosidase can include asubstrate that generates a fluorogenic product in response to theinteraction, a non-limiting example of which includes4-methylumbelliferyl-β-D-galactosidase.

In an aspect, the system further includes one or more reaction reagentsfor performing the multiplex analysis. In an aspect, the one or morereaction reagents include one or more reagents suitable for performingan amplification reaction. For example, the one or more reactionreagents can include one or more reagents for performing polymerasechain reaction (PCR) amplification such as, e.g., a DNA polymerase(e.g., Taq polymerase), deoxynucleoside triphosphates or deoxynucleotidetriphosphates (dNTPs), a buffer solution, bivalent cations (e.g.,magnesium or manganese ions), and monovalent cations (e.g., potassiumions). In an aspect, the one or more reaction reagents comprise one ormore reagents suitable for performing isothermal amplification. Forexample, the one or more reaction reagents can include reagents forperforming recombinase polymerase amplification (RPA), loop-mediatedisothermal amplification (LAMP), nucleic acid sequence basedamplification (NASBA), helicase-dependent amplification (HAD), andnicking enzyme amplification reaction (NEAR). For example, the one ormore reaction reagents can include one or more of a recombinase, asingle-stranded DNA-binding protein, a strand displacing polymerase, ahelicase, an endonuclease.

In an aspect, the one or more reaction reagents include one or morecomponents of a growth or culture medium. For example, the one or morereaction reagents can include components of a growth or culture mediumfor culturing cells and can be either synthetic or chemically defined ornon-synthetic or chemically undefined. The culture or growth medium canbe selected or designed to support growth of microorganisms (e.g.,bacteria or fungi) or cells (tissue culture cells or primary cells) andmay include a carbon source (e.g., glucose), various salts, and a sourceof amino acids and nitrogen. In some embodiments, the culture or growthmedium includes a selective medium for selective growth of a type(s) ofcell. In some embodiments, the culture or growth medium includes adifferential or indicator medium for detecting selective growth of atype(s) of cell. The one or more reaction reagents can include any of anumber of nutrients, amino acids, lipids, carbohydrates, sugars, andsalts necessary to promote growth of a given cell type(s). In someembodiments, the one or more reaction reagents include one or morereagents for lysing cells. For example, the reaction reagents caninclude enzymes (e.g., lysozyme) and detergents (e.g., NP-40, sodiumdeoxycholate).

In an aspect, the one or more reaction reagents include one or morereagents necessary or sufficient for performing a chemical reaction. Inan aspect, the one or more reaction reagents include one or morereagents necessary or sufficient for an enzymatic reaction. For example,the one or more reaction reagents can include enzymes, substrates,buffers, salts, ions, inorganic or organic co-factors, co-enzymes, andany other reagents necessary or sufficient for a given enzymaticreaction.

Described herein is a system for multiplexed detection of two or morenucleic acid sequences in a test sample. The system includes two or moresets of particles including a first set of particles, each particle ofthe first set of particles degradable in response to a firstenvironmental condition and having associated therewith a firstamplification primer set and a first optically detectable identifiercapable of emitting a first wavelength, the first amplification primerset selected to specifically interact with a first nucleic acidsequence, and the first optically detectable identifier indicative ofthe first amplification primer set; and at least one second set ofparticles, each particle of the at least second set of particlesdegradable in response to a second environmental condition and havingassociated therewith a second amplification primer set and a secondoptically detectable identifier capable of emitting a second wavelength,the second amplification primer set selected to specifically interactwith a second nucleic acid sequence, and the second optically detectableidentifier indicative of the second amplification primer set; and atleast one optically detectable reporter probe capable of constitutivelyemitting a third wavelength in response to amplification of the firstnucleic acid sequence in the test sample and/or the second nucleic acidsequence in the test sample.

FIG. 3 illustrates a non-limiting example of a system for multiplexeddetection of two or more nucleic acid sequences in a test sample. System300 includes two or more sets of particles 305 and at least oneoptically detectable reporter probe 330. The two or more sets ofparticles include a first set of particles 310 and at least one secondset of particles 320. Particles 312 a, 312 b, 312 c, 312 d, 312 e, and312 f are representative of particles in the first set of particles 310and are degradable in response to a first environmental condition (e.g.,temperature, pH, chemical reaction, electric field, or electromagneticenergy). Each of the particles 312 a-312f include a first amplificationprimer set 314 and a first optically detectable identifier 316. Thefirst amplification primer set 314 is selected to specifically interactwith a first nucleic acid sequence. First optically detectableidentifier 316 is capable of emitting a first wavelength (as representedby the horizontal line pattern in particles 312 a-312f ) and isindicative of the first amplification primer set. Particles 322 a, 322b, 322 c, 322 d, 322 e, and 322 f are representative of particles in theat least one second set of particles and are degradable in response to asecond environmental condition. In some embodiments, the secondenvironmental condition is the same as the first environmentalcondition. In other embodiments, the second environmental conditiondiffers from the first environmental condition. Each of the particles322 a-322f include a second amplification primer set 324 and a secondoptically detectable identifier 326. Second amplification primer set 324is selected to specifically interact with a second nucleic acidsequence. Second optically detectable identifier 326 is capable ofemitting a second wavelength (represented by the diagonal line patternin particles 322 a-322f ) and is indicative of the second amplificationprimer set. The first amplification primer set 314 and the secondamplification primer set 324 have sequence(s) selected or designed tohybridize to their respective first and second target nucleic acidsequences. For example, the amplification primer sets can includecomplimentary nuclei acid sequences that at least partially hybridize toa corresponding target nucleic acid sequence. At least one opticallydetectable reporter probe 330 is capable of constitutively (as shown byarrow 332) emitting a third wavelength 334 (represented by thecross-hatched lines) in response to amplification of the first nucleicacid sequence in the test sample by the first amplification primer set314 and/or the second nucleic acid sequence in the test sample by thesecond amplification primer set 324.

In an aspect, system 300 for multiplexed detection of two or morenucleic acid sequences in a test sample includes components designed todetect two or more nucleic acid sequences derived from one or more typesof bacteria. In an aspect, the first amplification primer set 314 andthe second amplification primer set 324 are selected to specificallyinteract with nucleic acid sequences derived from one or more types ofbacteria. For example, the first amplification primer set can havesequence(s) selected or designed to specifically interact (e.g., atleast partially hybridize) with a first nucleic acid sequence derivedfrom a bacteria type and the second amplification primer set can havesequence(s) selected or designed to specifically interact (e.g., atleast partially hybridize) with a second nucleic acid sequence derivedfrom the same bacteria type or a second bacteria type. For example, theamplification primer sets can have sequence(s) selected or designed tospecifically interact (e.g., at least partially hybridize) with nucleicacid sequences derived from bacteria and/or other microorganismssuspected of contaminating a water source, e.g., a well or reservoir.For example, the amplification primer sets can have sequence(s) selectedor designed to specifically interact (e.g., at least partiallyhybridize) with nucleic acid sequences derived from a sputum samplesuspected of containing Mycobacterium tuberculosis and/or other forms ofMycobacterium. For example, the amplification primer sets can havesequence(s) selected or designed to specifically interact (e.g., atleast partially hybridize) with nucleic acid sequences derived from ablood sample of an individual diagnosed with sepsis. In an aspect, theamplification primer sets can have sequence(s) selected or designed tospecifically interact (e.g., at least partially hybridize) with nucleicacid sequences associated with and/or derived from at least one ofmethicillin resistant Staphylococcus aureus, methicillin susceptibleStaphylococcus aureus, Escherichia coli, Streptococcus pneumonia,Pseudomonas aeruginosa, Staphylococcus epidermidis, Salmonella enterica,Klebsiella pneumonia, Streptococcus pyogenes, Acinetobacter baumannii,or Enterococcus faecalis.

In an aspect, system 300 for multiplexed detection of two or morenucleic acid sequences in a test sample includes components designed todetect two or more nucleic acid sequences derived from one or more celltypes. In an aspect, the first amplification primer set 314 and thesecond amplification primer set 324 of system 300 are selected tospecifically interact with nucleic acid sequences derived from one ormore cell types. For example, the amplification primer sets can havesequence(s) selected or designed to specifically interact (e.g., atleast partially hybridize) with two or more nucleic acid sequencesderived from microorganisms, e.g., bacteria, fungi, viruses, parasites,and the like. For example, the amplification primer sets can havesequence(s) selected or designed to specifically interact (e.g., atleast partially hybridize) with two or more nucleic acid sequencesderived from pathogens. For example, the amplification primer sets canhave sequence(s) selected or designed to specifically interact (e.g., atleast partially hybridize) with two or more nucleic acid sequencesderived from a body fluid or tissue. For example, the amplificationprimer sets can have sequence(s) selected or designed to specificallyinteract (e.g., at least partially hybridize) with two or more nucleicacid sequences derived from blood cells (e.g., red blood cells, whiteblood cells, platelets), inflammatory cells (e.g., macrophage, T-cells,B-cells, mast cells, eosinophils, basophils, neutrophils, monocytes),epithelial cells, hormone secreting cells, nerve cells, adipocytes,renal cells, contractile cells, and/or germ cells). In some embodiments,the amplification primer sets can have sequence(s) selected or designedto specifically interact (e.g., at least partially hybridize) with twoor more nucleic acid sequences derived from a tumor, malignant, and/orcancerous cell.

In an aspect, at least one of the two or more nucleic acid sequences inthe test sample comprises a single stranded DNA sequence, a doublestranded DNA sequence, or an RNA sequence. In an aspect, the firstamplification primer set 314 and the second amplification primer set 324of system 300 are selected to specifically interact with at least one ofsingle stranded DNA sequences, double stranded DNA sequences, or RNAsequences. In an aspect, the amplification primer sets have sequence(s)selected or designed to detect and/or amplify single or double strandedDNA or RNA sequence. In some embodiments amplification is done to detectthe presence of a particular nucleic acid sequence as an indicator ofthe presence of a particular cell type. In some embodiments,amplification is done to quantify the amount of a particular nucleicacid sequence. Each amplification primer set can include from one to tenshort oligonucleotide primers having from 10-40 bases each. In anaspect, the first amplification primer set 314 and the secondamplification primer set 324 are designed for use with polymerase chainreaction (PCR) amplification. In an aspect, the first amplificationprimer set 314 and the second amplification primer set 324 are designedfor real-time or quantitative PCR. For example, each amplificationprimer set can include a pair of DNA primers or oligonucleotides thatare complementary to the three prime ends of each of the sense andanti-sense strands of the target nucleic acid sequence. For example,each amplification primer set can include a forward primer and a reverseprimer.

In an aspect, the first amplification primer set 314 and the secondamplification primer set 324 are designed for use with an isothermalamplification process. For example, each amplification primer set caninclude from one to four pairs of DNA primers or oligonucleotides having10-40 bases each. For example, the amplification primer set can includeone or more loop primers. For example, the amplification primer set caninclude two or more inner primers and two or more outer primers. Forexample, the amplification primer set can include one or more forwardprimers and one or more reverse primers. For example, the firstamplification primer set and the second amplification primer set can bedesigned for one or more of recombinase polymerase amplification (RPA),loop-mediated isothermal amplification (LAMP), nucleic acid sequencebased amplification (NASBA), helicase-dependent amplification (HAD), ornicking enzyme amplification reaction (NEAR).

The first set of particles 310 are degradable in response to a firstenvironmental condition and the at least one second set of particles 320are degradable in response to a second environmental condition. In anaspect, the first environmental condition and the second environmentalcondition comprise at least one of temperature, pH, chemical reaction,electric field, or electromagnetic energy. For example, the first set ofparticles and the second set of particles are degradable in response toa change in at least one of temperature, pH, chemical reaction, electricfield, or electromagnetic energy. For example, the first set ofparticles and the second set of particles can be formed from materialsthat are degradable in response to a change in at least one oftemperature, pH, chemical reaction, electric field, or electromagneticenergy.

In some embodiments, the first environmental condition and the secondenvironmental condition are identical environmental conditions. Forexample, the first set of particles and the at least one second set ofparticles can be degradable in response to identical environmentalconditions, for example, an identical condition of temperature, pH,chemical reaction, electric field, and/or electromagnetic energy. Forexample, the first set of particles and the at least one second set ofparticles can be formed from materials that are degradable in responseto identical environmental conditions of temperature, pH, chemicalreaction, electric field, and/or electromagnetic energy.

In an aspect, the particles of the first set of particles 310 and of theat least one second set of particles 320 include a structure formed froma degradable material that is degradable in response to at least thefirst environmental condition or the second environmental condition. Inan aspect, the particles of the first set of particles 310 and of the atleast one second set of particles 320 include a structure formed from adegradable gel. In an aspect, the particles of the first set ofparticles 310 and of the at least one second set of particles 320include a structure formed from low melt agarose. In an aspect, theparticles of the first set of particles 310 and of the at least onesecond set of particles 320 include a structure formed from a degradablealginate. In an aspect, the particles of the first set of particles 310and of the at least one second set of particles 320 include a structureformed from a degradable sugar. In an aspect, the particles of the firstset of particles 310 and of the at least one second set of particles 320include a structure formed from a temperature-responsive degradablematerial. In an aspect, the particles of the first set of particles 310and of the at least one second set of particles 320 include a structureformed from a pH-responsive degradable material. In an aspect, theparticles of the first set of particles 310 and of the at least onesecond set of particles 320 include a structure formed from achemically-responsive degradable material. In an aspect, the particlesof the first set of particles 310 and of the at least one second set ofparticles 320 include a structure formed from an electricfield-responsive degradable material. In an aspect, the particles of thefirst set of particles 310 and of the at least one second set ofparticles 320 include a structure formed from an electromagneticenergy-responsive degradable material. Non-limiting examples ofenvironmental condition-responsive degradable materials for forming theparticles are presented above herein.

The two or more sets of particles 305 are designed for use in amultiplex analysis of two or more nucleic acid sequences in a testsample. In some embodiments, the multiplex analysis includes performingreactions in aqueous-in-oil droplets or an emulsion system. In anaspect, at least a portion of the particles of the first set ofparticles 310 and of the at least one second set of particles 320 aredistributable into an aqueous portion of aqueous-in-oil droplets. In anaspect, the particles of the first set of particles 310 and of the atleast one second set of particles 320 are hydrophilic. In an aspect, theparticles of the first set of particles 310 and of the at least onesecond set of particles 320 include a structure at least partiallyformed from a hydrophilic material. Non-limiting examples of materialsfor forming hydrophilic particles have been described above herein.

System 300 for multiplexed detection of two or more nucleic acidsequences in a test sample includes a first set of particles 310including a first optically detectable identifier 316 capable ofemitting a first wavelength, at least one second set of particles 320including a second optically detectable identifier 326 capable ofemitting a second wavelength, and at least one optically detectablereporter probe 330 capable of constitutively emitting a thirdwavelength. In an aspect, the first wavelength emitted by the firstoptically detectable identifier 316 is a first detectable color, thesecond wavelength emitted by the second optically detectable identifier326 is a second detectable color, and the third wavelengthconstitutively emitted by the at least one optically detectable reporterprobe 330 is a third detectable color. In an aspect, the firstwavelength, the second wavelength, and the third wavelength areoptically discernable from one another. In an aspect, the firstwavelength, the second wavelength, or the third wavelength is anultraviolet wavelength or wavelength band of electromagnetic energy. Inan aspect, at least one of the first wavelength, the second wavelength,or the third wavelength is a visible wavelength or wavelength band ofelectromagnetic energy. In an aspect, the first wavelength, the secondwavelength, or the third wavelength is a near infrared wavelength orwavelength band of electromagnetic energy.

In an aspect, the first optically detectable identifier 316 is a firstcolored dye or pigment and the second optically detectable identifier326 is a second colored dye or pigment. In an aspect, the firstoptically detectable identifier 316 includes a first fluorophore capableof emitting fluorescence at the first wavelength and the secondoptically detectable identifier 326 includes a second fluorophorecapable of emitting fluorescence at the second wavelength. In an aspect,the first optically detectable identifier 316 is a first quantum dotcapable of emitting the first wavelength and the second opticallydetectable identifier 326 is a second quantum dot capable of emittingthe second wavelength. Non-limiting examples of dyes, pigments,fluorescent dyes/fluorophores, colored or fluorescent particles, andquantum dots have been described above.

System 300 for multiplexed detection of two or more nucleic acidsequences in a test sample includes at least one optically detectablereporter probe 330 capable of constitutively emitting a third wavelengthin response to amplification of the first nucleic acid sequence in thetest sample and/or the second nucleic acid sequence in the test sample.In some embodiments, the at least one optically detectable reporterprobe 330 is associated with each particle of the first set of particles310 and with each particle of the at least one second set of particles320. In some embodiments, the at least one optically detectable reporterprobe 330 includes a first optically detectable reporter probe specificfor amplification of the first nucleic acid sequence and a secondoptically detectable reporter probe specific for amplification of thesecond nucleic acid sequence. For example, the first opticallydetectable reporter probe can be designed to constitutively emit adetectable wavelength in response to binding or hybridizing to the firstnucleic acid sequence while the second optically detectable reporterprobe can be designed to constitutively emit a second wavelength inresponse to binding or hybridizing to the second nucleic acid sequence.For example, the first optically detectable reporter probe and thesecond detectable reporter probe can include sequence specificTaqMan-like probes or molecular beacons.

In an aspect, the at least one optically detectable reporter probe 330comprises a donor-acceptor pair capable of constitutively emitting thethird wavelength. In an aspect, the at least one optically detectablereporter probe 330 comprises a fluorophore-quencher pair capable ofconstitutively emitting the third wavelength. For example, the opticallydetectable reporter probe can include a fluorophore-quencher pair inwhich a quencher molecule quenches fluorescence emitted by thefluorophore as long as the pair are in close proximity to one another.In some embodiments, the fluorophore-quencher pair are located on thesame molecule and changes in the configuration or conformation of themolecule upon interacting with a target changes the distance between thefluorophore and quencher. In some embodiments, the fluorophore and thequencher are located on separate binding molecules (e.g.,oligonucleotides, aptamers, or antibodies,) such that if the targets ofa first binding molecule and a second binding molecule are in closeproximity, binding of these molecules to their respective targets willbring the fluorophore and quencher into proximity of one another,leading to a decrease in detected fluorescence.

In some embodiments, the at least one optically detectable reporterprobe 330 includes a fluorophore-quencher pair that constitutively emitsa wavelength in response to a cleavage reaction triggered by theamplification of the first or the second nucleic acid sequence. In anaspect, the at least one optically detectable reporter probe 330includes at least one oligonucleotide probe with a cleavablefluorophore-quencher pair. For example, the at least one opticallydetectable reporter probe can include at least one TaqManTm probedesigned to anneal with a portion of a nucleic acid sequence to beamplified by a specific primer set and to release the fluorophore inresponse to exonuclease activity of a DNA polymerase (from Thermo FisherScientific, Waltham, Mass.). Other commercially available probe systemsthat constitutively emit a wavelength or wavelength band in response tointeracting with a nucleic acid sequence during an amplificationreaction include, but are not limited to, LightCycler® Probes andScorpions® Probes (from Sigma-Aldrich, Corp. St. Louis, Mo.) and MGBEclipse® Probes (from Integrated DNA Technologies, Skokie, Ill.).

In some embodiments, the at least one optically detectable reporterprobe 330 includes a fluorophore-quencher pair that constitutively emitsa wavelength in response to a structural confirmation change in theprobe. In an aspect, the at least one optically detectable reporterprobe 330 includes a molecular beacon. For example, the at least oneoptically detectable reporter probe can include at least oneoligonucleotide sequence with a stem-loop structure in the absence ofbinding, e.g., hybridizing, to a target and having a 5′ fluorophore anda 3′ quencher. Upon binding to a target nucleic acid sequence, thestem-loop structure is disrupted, the fluorophore and the quencherbecome spatially separated, resulting in emission of detectablefluorescence from the fluorophore.

In an aspect, the at least one optically detectable reporter probe 330is an RNA or DNA oligonucleotide-based aptamer that includes afluorophore-quencher pair. In an aspect, the at least one opticallydetectable reporter probe 330 includes an antibody or other bindingentity including a fluorophore-quencher pair. Non-limiting examples offluorophore-quencher pairs have been described above herein.

In an aspect, the at least one optically detectable reporter probe 330comprises a DNA intercalating agent capable of constitutively emittingthe third wavelength. For example, the optically detectable reporterprobe can include any of a number of fluorescing DNA-binding agents thatpreferentially bind to double stranded DNA and can be used as aquantification of amplification of a nucleic acid sequence. Non-limitingexamples include ethidium bromide, propidium iodide, DAPI,SYTO-9,SYTO-13, SYTO-82, SYBR Green 1, SYBR Gold, EvaGreen, and acridineorange.

In some embodiments, system 300 for multiplexed detection of two or morenucleic acid sequences further includes one or more reaction reagentsfor performing the multiplexed detection of the two or more nucleic acidsequences in the test sample. In an aspect, the one or more reactionreagents comprise one or more reaction reagents for amplification of thetwo or more nucleic acid sequences in the test sample. For example, theone or more reaction reagents can include one or more reagents forperforming polymerase chain reaction (PCR) amplification such as, e.g.,a DNA polymerase (e.g., Taq polymerase), deoxynucleoside triphosphatesor deoxynucleotide triphosphates (dNTPs), a buffer solution, bivalentcations (e.g., magnesium or manganese ions), and monovalent cations(e.g., potassium ions). In an aspect, the one or more reaction reagentscomprise one or more reagents suitable for performing isothermalamplification. For example, the one or more reaction reagents caninclude reagents for performing recombinase polymerase amplification(RPA), loop-mediated isothermal amplification (LAMP), nucleic acidsequence based amplification (NASBA), helicase-dependent amplification(HAD), and nicking enzyme amplification reaction (NEAR). For example,the one or more reaction reagents can include, but are not limited to,one or more of a reverse transcriptase, an RNase, an RNA polymerase, aligase, a recombinase, a single-stranded DNA-binding protein, a stranddisplacing polymerase, a helicase, an endonuclease.

In an aspect, system 300 for multiplexed analysis of two or more nucleicacid sequences in a test sample includes more than two sets ofparticles. In an aspect, system 300 includes three, four, five, six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen,sixteen, seventeen, eighteen, nineteen, or twenty sets of particles,each set of particles includes an amplification primer set and anoptically detectable identifier.

In an aspect, system 300 includes at least one third set of particles,each particle of the at least one third set of particles degradable inresponse to at least one of the first, the second, or a thirdenvironmental condition and having associated therewith a thirdamplification primer set and a third optically detectable identifiercapable of emitting a fourth wavelength, the third amplification primerset selected to specifically interact with a third nucleic acidsequence, and the third optically detectable identifier indicative ofthe third amplification primer set; and wherein the at least oneoptically detectable reporter probe is capable of constitutivelyemitting the third wavelength in response to amplification of the firstnucleic acid sequence in the test sample, the second nucleic acidsequence in the test sample, and/or the third nucleic acid sequence inthe test sample; and wherein the first wavelength, the secondwavelength, the third wavelength, and the fourth wavelength areoptically discernable from one another. In an aspect, the first, second,and third environmental condition is the same environmental condition(e.g., temperature, pH, chemical reaction, electric field, orelectromagnetic energy).

Described herein is a system for multiplexed detection of two or morebacterial nucleic acid sequences in a test sample. The system includestwo or more sets of particles including a first set of particles, eachparticle of the first set of particles degradable in response to a firsttemperature condition and having associated therewith a firstamplification primer set and a first fluorescent identifier capable ofemitting at a first wavelength, the first amplification primer setselected to specifically interact with a first bacterial nucleic acidsequence, and the first fluorescent identifier indicative of the firstamplification primer set; and at least one second set of particles, eachparticle of the at least one second set of particles degradable inresponse to a second temperature condition and having associatedtherewith a second amplification primer set and a second fluorescentidentifier capable of emitting at a second wavelength, the secondamplification primer set selected to specifically interact with a secondbacterial nucleic acid sequence, and the second fluorescent identifierindicative of the second amplification primer set; and at least onefluorescent intercalating agent capable of constitutively emitting at athird wavelength in response to amplification of the first bacterialnucleic acid sequence in the test sample and/or the second bacterialnucleic acid sequence in the test sample.

FIG. 4 illustrates a non-limiting example of a system for multiplexeddetection of two or more bacterial nucleic acid sequences in a testsample. System 400 includes two or more sets of particles 405 and atleast one fluorescent intercalating agent 430. The two or more sets ofparticles include a first set of particles 410 and at least one secondset of particles 420. Particles 412 a, 412 b, 412 c, 412 d, 412 e, and412 f are representative of particles in the first set of particles 410and are degradable in response to a first temperature condition. Each ofthe particles 412 a-412f include a first amplification primer set 414and a first fluorescent identifier 416. The first amplification primerset 414 is selected to specifically interact with a first bacterialnucleic acid sequence. First fluorescent identifier 416 is capable ofemitting at a first wavelength (as represented by the diagonal linepattern in particles 412 a-412f ) and is indicative of the firstamplification primer set. Particles 422 a, 422 b, 422 c, 422 d, 422 e,and 422 f are representative of particles in the at least one second setof particles and are degradable in response to a second temperaturecondition. In some embodiments, the second temperature condition is thesame as the first temperature condition. For example, the first set ofparticles 410 and the at least one second set of particles 420 can beformed from a material(s) that degrades at a temperature encounteredduring amplification cycling. Each of the particles 422 a-422f include asecond amplification primer set 424 and a second fluorescent identifier426. Second amplification primer set 424 is selected to specificallyinteract with a second bacterial nucleic acid sequence. Secondfluorescent identifier 426 is capable of emitting at a second wavelength(represented by the vertical line pattern in particles 422 a-422f ) andis indicative of the second amplification primer set. The firstamplification primer set 414 and the second amplification primer set 424have sequence(s) selected or designed to hybridize to their respectivefirst and second target bacterial nucleic acid sequences. For example,the amplification primer sets can include complimentary nuclei acidsequences that at least partially hybridize to a corresponding targetbacterial nucleic acid sequence. At least one fluorescent intercalatingagent 430 is capable of constitutively (as shown by arrow 432) emittingat a third wavelength 434 (represented by the cross-hatched lines) inresponse to amplification of the first bacterial nucleic acid sequencein the test sample by the first amplification primer set 414 and/or thesecond bacterial nucleic acid sequence in the test sample by the secondamplification primer set 424.

System 400 includes components designed to detect two or more bacterialnucleic acid sequences derived from one or more types of bacteria. In anaspect, the first amplification primer set 414 and the secondamplification primer set 424 are selected to specifically interact withbacterial nucleic acid sequences derived from one or more types ofbacteria. For example, the first amplification primer set can havesequence(s) selected or designed to specifically interact (e.g., atleast partially hybridize) with a first bacterial nucleic acid sequencederived from a bacteria type and the second amplification primer set canhave sequence(s) selected or designed to specifically interact (e.g., atleast partially hybridize) with a second bacterial nucleic acid sequencederived from the same bacteria type or a second bacteria type. Forexample, the amplification primer sets can have sequence(s) selected ordesigned to specifically interact (e.g., at least partially hybridize)with bacterial nucleic acid sequences derived from bacteria suspected ofcontaminating a water source, e.g., a well or reservoir. For example,the amplification primer sets can have sequence(s) selected or designedto specifically interact (e.g., at least partially hybridize) withnucleic acid sequences derived from a sputum sample suspected ofcontaining Mycobacterium tuberculosis and/or other forms ofMycobacterium. For example, the amplification primer sets can havesequence(s) selected or designed to specifically interact (e.g., atleast partially hybridize) with bacterial nucleic acid sequences derivedfrom a blood sample or other biological sample of an individualdiagnosed with sepsis. In an aspect, the amplification primer sets canhave sequence(s) selected or designed to specifically interact (e.g., atleast partially hybridize) with bacterial nucleic acid sequencesassociated with and/or derived from at least one of methicillinresistant Staphylococcus aureus, methicillin susceptible Staphylococcusaureus, Escherichia coli, Streptococcus pneumonia, Pseudomonasaeruginosa, Staphylococcus epidermidis, Salmonella enterica, Klebsiellapneumonia, Streptococcus pyogenes, Acinetobacter baumannii, orEnterococcus faecalis.

In an aspect, at least one of the two or more nucleic acid sequences inthe test sample comprises a single stranded DNA sequence, a doublestranded DNA sequence, or an RNA sequence. In an aspect, the firstamplification primer set 414 and the second amplification primer set 424of system 400 are selected to specifically interact with at least one ofsingle stranded DNA sequences, double stranded DNA sequences, or RNAsequences. In an aspect, the amplification primer sets have sequence(s)selected or designed to detect and/or amplify single or double strandedDNA or RNA sequence derived from bacteria. In some embodimentsamplification is done to detect the presence of a particular bacterialnucleic acid sequence as an indicator of the presence of a particularbacteria type. In some embodiments, amplification is done to quantifythe amount of a particular bacterial nucleic acid sequence. Eachamplification primer set can include from one to ten shortoligonucleotide primers having from 10-40 bases each. In an aspect, thefirst amplification primer set 414 and the second amplification primerset 424 are designed for use with polymerase chain reaction (PCR)amplification. In an aspect, the first amplification primer set 414 andthe second amplification primer set 424 are designed for real-time orquantitative PCR. For example, each amplification primer set can includea pair of DNA primers or oligonucleotides that are complementary to thethree prime ends of each of the sense and anti-sense strands of thetarget bacterial nucleic acid sequence. For example, each amplificationprimer set can include a forward primer and a reverse primer.

In an aspect, first amplification primer set 414 and the secondamplification primer set 424 are designed for use with an isothermalamplification process. For example, each amplification primer set caninclude from one to four pairs of DNA primers or oligonucleotides having10-40 bases each. For example, the amplification primer set can includeone or more loop primers. For example, the amplification primer set caninclude two or more inner primers and two or more outer primers. Forexample, the amplification primer set can include one or more forwardprimers and one or more reverse primers. For example, the firstamplification primer set and the second amplification primer set can bedesigned for one or more of recombinase polymerase amplification (RPA),loop-mediated isothermal amplification (LAMP), nucleic acid sequencebased amplification (NASBA), helicase-dependent amplification (HAD), ornicking enzyme amplification reaction (NEAR).

The first set of particles 410 are degradable in response to a firsttemperature condition and the at least one second set of particles 420are degradable in response to a second temperature condition. In someembodiments, the first temperature condition and the second temperaturecondition are the same temperature condition. For example, the first setof particles and the at least one second set of particles can bedegradable in response to identical temperature conditions. For example,the first set of particles and the at least one second set of particlescan be formed from materials that are degradable in response toidentical temperature conditions.

In an aspect, the particles of the first set of particles 410 and of theat least one second set of particles 420 include a structure formed froma degradable material that is degradable in response to at least thefirst temperature condition or the second temperature condition. In anaspect, the particles of the first set of particles 410 and of the atleast one second set of particles 420 include a structure formed from adegradable gel that is degradable in response to at least the firsttemperature condition or the second temperature condition. In an aspect,the particles of the first set of particles 410 and of the at least onesecond set of particles 420 include a structure formed from low meltagarose that is degradable in response to at least the first temperaturecondition or the second temperature condition. In an aspect, theparticles of the first set of particles 410 and of the at least onesecond set of particles 420 include a structure formed from atemperature-responsive degradable material that is degradable inresponse to at least the first temperature condition or the secondtemperature condition. Non-limiting examples of temperaturecondition-responsive degradable materials for forming the particles arepresented above herein.

The two or more sets of particles 405 are designed for use in amultiplex analysis of two or more bacterial nucleic acid sequences in atest sample. In some embodiments, the multiplex analysis includesperforming reactions in aqueous-in-oil droplets or an emulsion system.In an aspect, at least a portion of the particles of the first set ofparticles 410 and of the at least one second set of particles 420 aredistributable into an aqueous portion of aqueous-in-oil droplets. In anaspect, the particles of the first set of particles 410 and of the atleast one second set of particles 420 are hydrophilic. In an aspect, theparticles of the first set of particles 410 and of the at least onesecond set of particles 420 include a structure at least partiallyformed from a hydrophilic material. Non-limiting examples of materialsfor forming hydrophilic particles have been described above herein.

System 400 for multiplexed detection of two or more bacterial nucleicacid sequences in a test sample includes a first set of particles 410including a first fluorescent identifier 416 capable of emitting at afirst wavelength, at least one second set of particles 420 including asecond fluorescent identifier 426 capable of emitting at a secondwavelength, and at least one fluorescent intercalating agent 430 capableof constitutively emitting at a third wavelength. In an aspect, thefirst wavelength emitted by the first fluorescent identifier 416 is afirst detectable color, the second wavelength emitted by the secondfluorescent identifier 426 is a second detectable color, and the thirdwavelength constitutively emitted by the at least one fluorescentintercalating agent 430 is a third detectable color. In an aspect, thefirst wavelength, the second wavelength, and the third wavelength areoptically discernable from one another. In an aspect, the firstwavelength, the second wavelength, or the third wavelength is anultraviolet wavelength or wavelength band of electromagnetic energy. Inan aspect, at least one of the first wavelength, the second wavelength,or the third wavelength is a visible wavelength or wavelength band ofelectromagnetic energy. In an aspect, the first wavelength, the secondwavelength, or the third wavelength is a near infrared wavelength orwavelength band of electromagnetic energy.

In an aspect, the first fluorescent identifier 416 is a firstfluorescent dye or pigment and the second fluorescent identifier 426 isa second fluorescent dye or pigment. In an aspect, the first fluorescentidentifier 416 includes a first fluorophore capable of emittingfluorescence at the first wavelength and the second fluorescentidentifier 426 includes a second fluorophore capable of emittingfluorescence at the second wavelength. In an aspect, the firstfluorescent identifier 416 is a first quantum dot capable of emittingthe first wavelength and the second fluorescent identifier 426 is asecond quantum dot capable of emitting the second wavelength.Non-limiting examples of dyes, pigments, fluorescent dyes/fluorophores,colored or fluorescent particles, and quantum dots have been describedabove.

System 400 for multiplexed detection of two or more bacterial nucleicacid sequences in a test sample includes at least one fluorescentintercalating agent 430 capable of constitutively emitting at a thirdwavelength in response to amplification of the first bacterial nucleicacid sequence in the test sample and/or the second bacterial nucleicacid sequence in the test sample. In some embodiments, the at least onefluorescent intercalating agent 430 is associated with each particle ofthe first set of particles 410 and with each particle of the at leastone second set of particles 420.

In an aspect, the fluorescent intercalating agent 430 includes afluorescent DNA intercalating agent. For example, the fluorescentintercalating agent can include any of a number of fluorescingDNA-binding agents that preferentially bind to double stranded DNA andcan be used as an optically detectable reporter probe of amplificationof a nucleic acid sequence. Non-limiting examples of fluorescentintercalating agents include, but are not limited to, ethidium bromide,propidium iodide, DAPI, SYTO-9, SYTO-13, SYTO-82, SYBR Green 1, SYBRGold, EvaGreen, and acridine orange.

In some embodiments, system 400 may further include other opticallydetectable reporter probes capable of constitutively emitting at thethird wavelength or at a fourth wavelength in response to amplificationof the first bacterial nucleic acid sequence in the test sample and/orthe second bacterial nucleic acid sequence in the test sample, a capableof constitutively emitting the third wavelength. Non-limiting examplesinclude a donor-acceptor pair or fluorophore-quencher pair capable ofconstitutively emitting at the third wavelength or at a fourthwavelength in response to an amplification reaction and have beendescribed above herein.

In some embodiments, system 400 for multiplexed detection of two or morebacterial nucleic acid sequences further includes one or more reactionreagents for performing the multiplexed detection of the two or morebacterial nucleic acid sequences in the test sample. In an aspect, theone or more reaction reagents include one or more reaction reagents foramplification of the two or more bacterial nucleic acid sequences in thetest sample. For example, the one or more reaction reagents can includeone or more reagents for performing polymerase chain reaction (PCR)amplification such as, e.g., a DNA polymerase (e.g., Taq polymerase),deoxynucleoside triphosphates or deoxynucleotide triphosphates (dNTPs),a buffer solution, bivalent cations (e.g., magnesium or manganese ions),and monovalent cations (e.g., potassium ions). In an aspect, the one ormore reaction reagents comprise one or more reagents suitable forperforming isothermal amplification. For example, the one or morereaction reagents can include reagents for performing recombinasepolymerase amplification (RPA), loop-mediated isothermal amplification(LAMP), nucleic acid sequence based amplification (NASBA),helicase-dependent amplification (HAD), and nicking enzyme amplificationreaction (NEAR). For example, the one or more reaction reagents caninclude, but are not limited to, one or more of a reverse transcriptase,an RNase, an RNA polymerase, a ligase, a recombinase, a single-strandedDNA-binding protein, a strand displacing polymerase, a helicase, anendonuclease. In some embodiments, the one or more reaction reagentsinclude one or more reagents for lysing bactera. For example, thereaction reagents can include enzymes (e.g., lysozyme, lysostaphin) anddetergents (e.g., NP-40, sodium deoxycholate).

In an aspect, system 400 for multiplexed analysis of two or morebacterial nucleic acid sequences in a test sample includes more than twosets of particles. In an aspect, system 400 includes three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen, seventeen, eighteen, nineteen, or twenty sets ofparticles, each set of particles includes an amplification primer setand a fluorescent identifier.

In an aspect, system 400 includes at least one third set of particles,each particle of the at least one third set of particles degradable inresponse to at least one of the first, the second, or a thirdtemperature condition and having associated therewith a thirdamplification primer set and a third fluorescent identifier capable ofemitting at a fourth wavelength, the third amplification primer setselected to specifically interact with a third bacterial nucleic acidsequence, and the third fluorescent identifier indicative of the thirdamplification primer set; and wherein the at least one fluorescentintercalating agent is capable of constitutively emitting at the thirdwavelength in response to amplification of the first bacterial nucleicacid sequence in the test sample, the second bacterial nucleic acidsequence in the test sample, and/or the third bacterial nucleic acidsequence in the test sample; and wherein the first wavelength, thesecond wavelength, the third wavelength, and the fourth wavelength areoptically discernable from one another. In an aspect, the first, second,and third temperature condition is the same temperature condition.

Described herein is a system for multiplexed analysis of antibioticresistance in a bacterial sample. The system includes two or more setsof particles including a first set of particles, each particle of thefirst set of particles degradable in response to a first environmentalcondition and having associated therewith a first antibiotic and a firstoptically detectable identifier capable of emitting a first wavelength,the first antibiotic having at least one of bactericidal orbacteriostatic activity against a first subset of bacteria, and thefirst optically detectable identifier indicative of the firstantibiotic; and at least one second set of particles, each particle ofthe at least second set of particles degradable in response to a secondenvironmental condition and having associated therewith a secondantibiotic and a second optically detectable identifier capable ofemitting a second wavelength, the second antibiotic having at least oneof bactericidal or bacteriostatic activity against a second subset ofbacteria, and the second optically detectable identifier indicative ofthe second antibiotic; and at least one optically detectable reporterprobe capable of constitutively emitting a third wavelength in responseto viability of bacteria in the bacterial sample.

FIG. 5 illustrates a non-limiting example of a system 500 formultiplexed analysis of antibiotic resistance in a bacterial sample.System 500 includes two or more sets of particles 505 and at least oneoptically detectable reporter probe 530. The two or more sets ofparticles 505 include a first set of particles 510 and at least onesecond set of particles 520. Particles 512 a, 512 b, 512 c, 512 d, 512e, and 512 f are representative of particles in the first set ofparticles 510 and are degradable in response to a first environmentalcondition (e.g., temperature, pH, chemical reaction, electric field, orelectromagnetic energy). Each of the particles 512 a-512 f include afirst antibiotic 514 and a first optically detectable identifier 516.The first antibiotic 514 has at least one of bactericidal orbacteriostatic activity against a first subset of bacteria. Firstoptically detectable identifier 516 is capable of emitting a firstwavelength (as represented by the diagonal lines in particles 512 a-512f ) and is indicative of the first antibiotic 514. Particles 522 a, 522b, 522 c, 522 d, 522 e, and 522 f are representative of particles in theat least one second set of particles 520 and are degradable in responseto a second environmental condition. In some embodiments, the secondenvironmental condition is the same as the first environmentalcondition. In other embodiments, the second environmental conditiondiffers from the first environmental condition. Each of the particles522 a-522 f includes a second antibiotic 524 and a second opticallydetectable identifier 526. Second antibiotic 524 has at least one ofbactericidal or bacteriostatic activity against a second subset ofbacteria. In some embodiments, the second subset of bacteria differsfrom first subset of bacteria. In other embodiments, the second subsetof bacteria is identical to the first subset of bacteria. For example,system 500 can be configured for multiplexed analysis of bacterialresistance of a single subset or type of bacteria against multipleantibiotics, each antibiotic in its own set of particles with its ownoptically detectable identifier. Second optically detectable identifier526 is capable of emitting a second wavelength (represented by thevertical line pattern in particles 522 a-522 f ) and is indicative ofthe second antibiotic 524. At least one optically detectable reporterprobe 530 is capable of constitutively (as shown by arrow 532) emittinga third wavelength 534 (represented by the cross-hatched lines) inresponse to viability of bacteria in the bacterial sample.

System 500 is configured for multiplexed analysis of antibioticresistance in a bacterial sample. The bacterial sample can include asample taken from a subject, e.g., a mammalian subject, and can includea sample from a bodily fluid, a tissue, or a swab. Alternatively, thebacterial sample can include a sample taken from an environmentalsource, e.g., water, air, soil, a surface, food, beverage, medicine, orother component of the environment.

The two or more sets of particles 505 of system 500 for multiplexedanalysis of antibiotic resistance in a bacterial sample include a firstset of particles 510 including a first antibiotic 514 havingbactericidal or bacteriostatic activity against a first subset ofbacteria and at least one second set of particles 520 including secondantibiotic 524 having bactericidal or bacteriostatic activity against asecond subset of bacteria. In an aspect, the subsets of bacteria includesubsets of Gram positive bacteria, Gram negative bacteria, Mycobacteria,aerobic bacteria, anaerobic bacteria. In an aspect, a subset of bacteriaincludes a phylum of bacteria, a class of bacteria, an order ofbacteria, a family of bacteria, a genus of bacteria, a species ofbacteria, or a sub-species or strain of bacteria.

In an aspect, the first subset of bacteria and the second subset ofbacteria are different subsets of bacteria. For example, the firstsubset of bacteria and the second subset of bacteria are part of a testsample derived from a bodily fluid, e.g., urine, or an environmentalsample, e.g., a water sample. In an aspect, the first subset of bacteriaand the second subset of bacteria are identical subsets of bacteria. Forexample, the test sample can include a defined subset of bacteria foruse in the multiplexed analysis of antibiotic resistance. For example,the system for multiplexed analysis of antibiotic resistance can includetwo or more subsets of particles, each subset of particles including adifferent antibiotic for assessing antibiotic resistance against aspecific bacteria or subset of bacteria.

In an aspect, the first subset of bacteria or the second subset ofbacteria includes one or more strains of Escherichia coli. For example,the subset of bacteria can include one or more strains of Escherichiacoli associated with gastrointestinal infection (e.g., O157:H7, O104:H4,and O104:H21). For example, the subset of bacteria can include one ormore strains of Escherichia coli associated with urinary tract infection(e.g., uropathogenic E. coli).

In an aspect, the first subset of bacteria or the second subset ofbacteria includes one or more species of Mycobacterium. In an aspect,the first subset of bacteria or the second subset of bacteria includesMycobacterium tuberculosis. For example, the system can be designed formultiplexed analysis of antibiotic resistance of bacteria associatedwith tuberculosis. For example, the system can be designed formultiplexed analysis of antibiotic resistance against Mycobacteriumtuberculosis. For example, each set of the two or more sets of particlescan include a different antibiotic for testing against a samplecontaining strains of Mycobacterium tuberculosis or other diseasecausing Mycobacterium, non-limiting examples of antibiotic includingisoniazid, rifampicin, bedaquiline, delamanid, streptomycin,clofazimine, ethambutol, pyrazinamide, linezolid, fluoroquinolones,ethionamide, capreomycin, and para-aminosalicylic acid. In an aspect,the first subset of bacteria or the second subset of bacteria includesone or more bacteria or strains of bacteria from the Mycobacteriumtuberculosis complex (e.g., M. africanum, M. bovis BCG, M. tuberculosis,and M. canetti, to name just a few).

In an aspect, the first subset of bacteria and the second subset ofbacteria includes one or more of methicillin resistant Staphylococcusaureus, methicillin susceptible Staphylococcus aureus, Escherichia coli,Streptococcus pneumonia, Pseudomonas aeruginosa, Staphylococcusepidermidis, Salmonella enterica, Klebsiella pneumonia, Streptococcuspyogenes, Acinetobacter baumannii, or Enterococcus faecalis. Forexample, the system can be designed for multiplexed analysis ofantibiotic resistance of bacteria associated with sepsis.

System 500 for multiplexed analysis of antibiotic resistance in abacterial sample includes a first set of particles 510 degradable inresponse to a first environmental condition and a second set ofparticles 520 degradable in response to a second environmentalcondition. In an aspect, the first environmental condition and thesecond environmental condition comprise at least one of temperature, pH,chemical reaction, electric field, or electromagnetic energy. Forexample, the first set of particles and the at least one second set ofparticles can be degradable in response to a change in at least one oftemperature, pH, chemical reaction, electric field, or electromagneticenergy. For example, the first set of particles and the at least onesecond set of particles can be formed from materials that are degradablein response to a change in at least one of temperature, pH, chemicalreaction, electric field, or electromagnetic energy.

In some embodiments, the first environmental condition and the secondenvironmental condition are identical environmental conditions. Forexample, the first set of particles and the at least one second set ofparticles can be degradable in response to identical environmentalconditions, for example, an identical condition of temperature, pH,chemical reaction, electric field, and/or electromagnetic energy. Forexample, the first set of particles and the at least one second set ofparticles can be formed from materials that are degradable in responseto identical environmental conditions of temperature, pH, chemicalreaction, electric field, and/or electromagnetic energy.

In an aspect, the particles of the first set of particles 510 and of theat least one second set of particles 520 include a structure formed froma degradable material that is degradable in response to at least thefirst environmental condition or the second environmental condition. Inan aspect, the particles of the first set of particles 510 and of the atleast one second set of particles 520 include a structure formed from adegradable gel. In an aspect, the particles of the first set ofparticles 510 and of the at least one second set of particles 520include a structure formed from low melt agarose. In an aspect, theparticles of the first set of particles 510 and of the at least onesecond set of particles 520 include a structure formed from a degradablealginate. In an aspect, the particles of the first set of particles 510and of the at least one second set of particles 520 include a structureformed from a degradable sugar. In an aspect, the particles of the firstset of particles 510 and of the at least one second set of particles 520include a structure formed from a temperature-responsive degradablematerial. In an aspect, the particles of the first set of particles 510and of the at least one second set of particles 520 include a structureformed from a pH-responsive degradable material. In an aspect, theparticles of the first set of particles 510 and of the at least onesecond set of particles 520 include a structure formed from achemically-responsive degradable material. In an aspect, the particlesof the first set of particles 510 and of the at least one second set ofparticles 520 include a structure formed from an electricfield-responsive degradable material. In an aspect, the particles of thefirst set of particles 510 and of the at least one second set ofparticles 520 include a structure formed from an electromagneticenergy-responsive degradable material. Non-limiting examples ofenvironmental condition-responsive degradable materials for forming theparticles of the two or more sets of particles are presented aboveherein.

The two or more sets of particles 505 are designed or selected for usein a multiplex analysis of antibiotic resistance. In some embodiments,the multiplex analysis of antibiotic resistance includes performingreactions in aqueous-in-oil droplets or an emulsion system. In anaspect, at least a portion of the particles of the first set ofparticles 510 and of the at least one second set of particles 520 aredistributable into an aqueous portion of aqueous-in-oil droplets oremulsion. In an aspect, the particles of the first set of particles 510and of the at least one second set of particles 520 are hydrophilic. Inan aspect, the particles of the first set of particles 510 and of the atleast one second set of particles 520 include a structure at leastpartially formed from a hydrophilic material. Non-limiting exampleshydrophilic material for use in forming degradable particles have beendescribed above herein.

System 500 for multiplexed analysis of antibiotic resistance in abacterial sample includes two or more sets of particles 505 including afirst set of particles 510 including a first antibiotic 514 and at leastone second set of particles 520 including a second antibiotic 524. Thefirst antibiotic 514 and the second antibiotic 524 can include any of anumber of antibiotic reagents having bactericidal activity (i.e., killsbacteria) or bacteriostatic activity (i.e., inhibits bacterial growth).Non-limiting examples of antibiotics specific for Gram-negativebacteria, Gram-positive positive bacteria, Mycobacterium, and withnarrow spectrum or broad-spectrum of reactivity include aminoglycosides,ansamycins, carbapenems, aminoglycosides, clindamycin, trimethoprim,cephalosporins, glycopeptides, lincosamides, macrolides, beta-lactams,monobactams, tetracyclines, sulfonamides, quinolones, penicillins,nitrofurans, and oxazolidinones. In an aspect, the antibiotics caninclude any of a number of antibiotics used in the treatment oftuberculosis, non-limiting examples of which include capreomycin,cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide,rifampicin, rifabutin, rifapentine, and streptomycin. Other non-limitingexamples of antibiotics include chloramphenicol, fosfomycin, fusidicacid, metronidazole, thiamphenicol, and tigecycline. It is alsoanticipated that new antibiotics in development or not yet described canbe incorporated into the two or more sets of particles of the system.

System 500 for multiplexed analysis of antibiotic resistance in abacterial sample includes a first set of particles 510 including a firstoptically detectable identifier 516 capable of emitting a firstwavelength, at least one second set of particles 520 including a secondoptically detectable identifier 526 capable of emitting a secondwavelength, and at least one optically detectable reporter probe 530capable of constitutively emitting a third wavelength. In an aspect, thefirst wavelength emitted by the first optically detectable identifier516 is a first detectable color, the second wavelength emitted by thesecond optically detectable identifier 526 is a second detectable color,and the third wavelength constitutively emitted by the at least oneoptically detectable reporter probe 530 is a third detectable color. Inan aspect, the first wavelength, the second wavelength, and the thirdwavelength are optically discernable from one another. In an aspect, thefirst wavelength, the second wavelength, or the third wavelength is anultraviolet wavelength or wavelength band of electromagnetic energy. Inan aspect, at least one of the first wavelength, the second wavelength,or the third wavelength is a visible wavelength or wavelength band ofelectromagnetic energy. In an aspect, the first wavelength, the secondwavelength, or the third wavelength is a near infrared wavelength orwavelength band of electromagnetic energy.

In an aspect, the first optically detectable identifier 516 is a firstcolored dye or pigment and the second optically detectable identifier526 is a second colored dye or pigment. In an aspect, the firstoptically detectable identifier 516 is a first fluorophore capable ofemitting fluorescence at the first wavelength and the second opticallydetectable identifier 526 is a second fluorophore capable of emittingfluorescence at the second wavelength. In an aspect, the first opticallydetectable identifier 516 is a first quantum dot capable of emitting thefirst wavelength and the second optically detectable identifier 526 is asecond quantum dot capable of emitting the second wavelength.Non-limiting examples of dyes, pigments, fluorophores, colored andfluorescent particles, and quantum dots have been described above.

System 500 for multiplexed analysis of antibiotic resistance in abacterial sample includes at least one optically detectable reporterprobe 530 capable of constitutively emitting a third wavelength inresponse to viability of bacteria in the bacterial sample. In someembodiments, the at least one optically detectable reporter probe 530 isassociated with each particle of the first set of particles 510 and witheach particle of the at least one second set of particles 520. In someembodiments, the at least one optically detectable reporter probe 530includes a first optically detectable reporter probe specific forviability of the first subset of bacteria and a second opticallydetectable reporter probe specific for viability of the second subset ofbacteria. For example, a first optically detectable reporter probe caninclude a bacteriophage specific for the first subset of bacteria and asecond optically detectable reporter probe can include a secondbacteriophage specific for the second subset of bacteria, wherein eachbacteriophage type includes a luciferase reporter gene forconstitutively emitting the third wavelength in response to infectingtheir respective bacteria subsets. In this instance, the two differentreporter probes constitutively emit the same wavelength, i.e., the thirdwavelength, while the optically detectable identifiers provide anindication of which antibiotics are present in a given droplet.

In an aspect, the at least one optically detectable reporter probe 530comprises a donor-acceptor pair capable of constitutively emitting thethird wavelength. In an aspect, the at least one optically detectablereporter probe 530 comprises a fluorophore-quencher pair capable ofconstitutively emitting the third wavelength. For example, the opticallydetectable reporter probe can include a donor-acceptorpair/fluorophore-quencher pair capable or constitutively emitting thethird wavelength in response to viability of bacteria in the bacterialsample. For example, the optically detectable reporter probe can includea fluorophore-quencher pair in which the quencher molecule quenchesfluorescence emitted by the fluorophore as long as the pair are in closeproximity by the process of FRET. In an aspect, the at least oneoptically detectable reporter probe includes an antibody, aptamer, orother binding entity including a fluorophore-quencher pair. For example,the at least one optically detectable reporter probe can include anantibody with a fluorophore and a quencher in close proximity to oneanother which separate in response to either degradation of the antibodyupon cellular internalization or a conformational change in the antibodywhen it binds to its specific target. Non-limiting examples offluorophore-quencher pairs with antibodies, oligonucleotide, or aptamersubstrates have been described above herein.

In an aspect, the at least one optically detectable reporter probe 530comprises a DNA intercalating agent capable of constitutively emittingthe third wavelength. For example, the optically detectable reporterprobe can include a DNA intercalating agent capable of constitutivelyemitting the third wavelength in response to viability of bacteria inthe bacterial sample. Non-limiting examples include ethidium bromide,propidium iodide, DAPI, SYTO-9, SYTO-13, SYTO-82, SYBR Green 1, SYBRGold, EvaGreen, and acridine orange. In some embodiments, the DNAintercalating agent is capable of entering a viable bacterial cell. Insome embodiments, the DNA intercalating agent is only able to enter abacterial cell with a compromised cell membrane, i.e., a dead or dyingbacterial cell. In an aspect, the at least one optically detectablereporter probe 530 comprises a probe of membrane integrity capable ofconstitutively emitting the third wavelength. For example, the opticallydetectable reporter probe can include a DNA intercalating dye or vitaldye that is capable of entering and concentrating in bacteria once themembrane has become compromised as a result of cell death.

In an aspect, the at least one optically detectable reporter probe 530comprises a bacteriophage with a reporter gene. For example, theoptically detectable reporter probe can include a bacteriophage with areporter gene capable of constitutively emitting the third wavelength inresponse to viability of bacteria in the bacterial sample. For example,a reporter gene or genes can be incorporated into the phage genome andupon infection of bacteria, the reporter gene(s) carried by thebacteriophage is expressed. As such, bacteriophage including a reportergene can be used as an indicator of bacterial viability. In an aspect,the reporter probe is a bacteriophage with a broad spectrum of bacterialinfection. In an aspect, the reporter probe is a bacteriophage with anarrow spectrum of bacterial infection. Additional information regardingthe breadth of bacteriophage can be found on various database websites,non-limiting examples of which have been cited above herein.

In an aspect, the at least one optically detectable reporter probe 530comprises a bacteriophage including a reporter gene that generates acolorimetric, fluorescent, chemiluminescent, or bioluminescent signal inresponse to infection and propagation of viable bacterial cells. In someinstances, the response is spontaneous (e.g., autofluorescenceassociated with green fluorescent protein). In some instances, theresponse requires a substrate or cofactor, either endogenous orexogenous to the bacteria (e.g., when the reporter gene is an enzyme).Non-limiting examples of reporter genes include those encoding green,yellow or red fluorescent protein (GFP, YFP, or RFP, respectively),bacterial luciferase, firefly luciferase, beta-galactosidase (lacZ),chloramphenyl acetyltransferase (CAT), beta-glucuronidase (GUS). Forother non-limiting examples of reporting via reporter genes, see, e.g.,Smartt & Ripp (2011) Anal. Bioanal. Chem. 400:991-1007, which isincorporated herein by reference.

In an aspect, the at least one optically detectable reporter probe 530comprises a substrate capable of constitutively emitting the thirdwavelength in response to interaction with an enzyme. In an aspect, thesubstrate is at least one of a chemical substrate, a lipid-basedsubstrate, a peptide-based substrate, or a protein-based substrate. Inan aspect, the interaction of the substrate with a target enzymeproduces a colorimetric or fluorogenic product. In an aspect, theinteraction of the substrate with a target enzyme is detected bychemiluminescence. In an aspect, the at least one optically detectablereporter probe includes a substrate for at least one ofbeta-glucuronidase, beta-glucosidase, beta-galactosidase,beta-lactamase, beta-glucuronidase, alkaline phosphatase, luciferase,cytochrome P450, deubiquitinating enzyme, kinase, phosphatase, lipase,phospholipase, protease, or peptidase. In an aspect, the at least oneoptically detectable reporter probe includes a substrate that interactswith a component of a cell membrane (e.g., lipids, proteins and proteinreceptors, and carbohydrates). A number of substrates whichconstitutively emit a wavelength in response to interaction with anenzymatic target are available from commercial sources (from, e.g.,Thermo Fisher Scientific, Waltham, Mass.).

In an aspect, the at least one optically detectable reporter probe 530includes a substrate for beta-glucuronidase capable of constitutivelyemitting the third wavelength in response to viability of bacteria inthe bacterial sample. For example, the substrate for beta-glucuronidasecan include a substrate that generates a colorimetric precipitate inresponse to the interaction, non-limiting examples of which have beendescribed above herein. For example, the substrate forbeta-glucuronidase can include a substrate that generates a fluorogenicproduct in response to the interaction, a non-limiting example of which4-methylumbelliferyl-β-D-glucuronide (MUG).

In an aspect, the at least one optically detectable reporter probe 530includes a substrate for beta-galactosidase capable of constitutivelyemitting the third wavelength in response to viability of bacteria inthe bacterial sample. For example, the substrate for beta-galactosidasecan include a substrate that generates a colorimetric precipitate inresponse to the interaction, non-limiting examples of which have beendescribed above herein. For example, the substrate of beta-galactosidasecan include a substrate that generates a fluorogenic product in responseto the interaction, a non-limiting example of which includes4-methylumbelliferyl-β-D-galactosidase.

In some embodiments, system 500 for multiplexed analysis of antibioticresistance in a bacterial sample includes one or more reaction reagentsfor performing the multiplexed analysis. In an aspect, system 500includes one or more components of a culture medium for growingbacteria. For example, the one or more reaction reagents can includecomponents of a growth, nutrient, or culture medium for culturing cellsand can be either synthetic or chemically defined or non-synthetic orchemically undefined. The culture or growth medium can includecomponents to support growth of bacteria and may include a carbon source(e.g., glucose), various salts, and a source of amino acids andnitrogen. In some embodiments, the culture, nutrient, or growth mediumincludes a selective medium for selective growth of a type(s) of cell.In some embodiments, the culture, nutrient, or growth medium includes adifferential or indicator medium for detecting selective growth of atype(s) of cell. The one or more reaction reagents can include any of anumber of nutrients, amino acids, lipids, carbohydrates, sugars, andsalts necessary to promote growth of bacteria. The one or more reactionreagents can further include one or more reagents for lysing bacteria,e.g., lysozyme.

In an aspect, system 500 for multiplexed analysis of antibioticresistance in a bacterial sample includes more than two sets ofparticles. In an aspect, system 500 includes three, four, five, six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen,sixteen, seventeen, eighteen, nineteen, or twenty sets of particles,each set of particles includes an antibiotic and an optically detectableidentifier.

In an aspect, system 500 includes at least one third set of particles,each particle of the at least one third set of particles degradable inresponse to at least one of the first, the second, or a thirdenvironmental condition and having associated therewith a thirdantibiotic and a third optically detectable identifier capable ofemitting a fourth wavelength, the third antibiotic having at least oneof bactericidal or bacteriostatic activity against a third subset ofbacteria, and the third optically detectable identifier indicative ofthe third antibiotic; and wherein the at least one optically detectablereporter probe is capable of constitutively emitting the thirdwavelength in response to viability of bacteria in the bacterial sample;and wherein the first wavelength, the second wavelength, the thirdwavelength, and the fourth wavelength are discernable or distinguishablefrom one another. In an aspect, the first, second, and thirdenvironmental condition is the same environmental condition (e.g.,temperature, pH, chemical reaction, electric field, or electromagneticenergy). In an aspect, the third subset of bacteria is identical to thefirst and second subset of bacteria, such that a single subset ofbacteria is assessed for antibiotic resistance against the first,second, and the at least third antibiotic in the system.

Described herein is a system configured for multiplexed analysis of twoor more antigens in a test sample. In an embodiment, the system formultiplexed analysis of two or more antigens includes two or more setsof particles including a first set of particles, each particle of thefirst set of particles degradable in response to a first environmentalcondition and having associated therewith a first antibody set and afirst optically detectable identifier capable of emitting a firstwavelength, the first antibody set including two or more antibodiesspecific for proximal targets on a first antigen, wherein the two ormore antibodies of the first antibody set include modifications capableof interacting in an antibody-based proximity assay, and the firstoptically detectable identifier indicative of the first antibody set;and at least one second set of particles, each particle of the at leastone second set of particles degradable in response to a secondenvironmental condition and having associated therewith a secondantibody set and a second optically detectable identifier capable ofemitting a second wavelength, the second antibody set including two ormore antibodies specific for proximal targets on a second antigen,wherein the two or more antibodies of the second antibody set includemodifications capable of interacting in an antibody-based proximityassay, and the second optically detectable identifier indicative of thesecond antibody set; and at least one optically detectable reporterprobe capable of constitutively emitting a third wavelength in responseto the two or more antibodies of the first antibody set binding to theirproximal targets on the first antigen and/or the two or more antibodiesof the second antibody set binding to their proximal targets on thesecond antigen.

FIG. 6 illustrates a non-limiting example of a system for multiplexedanalysis of two or more antigens in a test sample. System 600 includestwo or more sets of particles 605 and at least one optically detectablereporter probe 630. The two or more sets of particles 605 include afirst set of particles 610 and at least one second set of particles 620.Particles 612 a, 612 b, 612 c, 612 d, 612 e, and 612 f arerepresentative of particles in the first set of particles 610 and aredegradable in response to a first environmental condition (e.g.,temperature, pH, chemical reaction, electric field, or electromagneticenergy). Each of the particles 612 a-612 f include a first antibody set614 and a first optically detectable identifier 616. The first antibodyset 614 includes two or more antibodies specific for proximal targets ona first antigen, wherein the two or more antibodies of the firstantibody set 614 include modifications capable of interacting in anantibody-based proximity assay. First optically detectable identifier616 is capable of emitting a first wavelength (as represented by thevertical line pattern in particles 612 a-612 f ) and is indicative ofthe first antibody set. Particles 622 a, 622 b, 622 c, 622 d, 622 e, and622 f are representative of particles in the at least one second set ofparticles 620 and are degradable in response to a second environmentalcondition. In some embodiments, the second environmental condition isthe same as the first environmental condition. In other embodiments, thesecond environmental condition differs from the first environmentalcondition. Each of the particles 622 a-622 f includes a second antibodyset 624 and a second optically detectable identifier 626. Secondantibody set 624 includes two or more antibodies specific for proximaltargets on a second antigen, wherein the two or more antibodies of thesecond antibody set 624 include modifications capable of interacting inan antibody-based proximity assay. Second optically detectableidentifier 626 is capable of emitting a second wavelength (representedby the horizontal line pattern in particles 622 a-622 f) and isindicative of the second antibody set. System 600 further includes atleast one optically detectable reporter probe 630 capable ofconstitutively (as shown by arrow 632) emitting a third wavelength 634(represented by the cross-hatched lines) in response to the two or moreantibodies of the first antibody set 614 binding to their proximaltargets on the first antigen and/or the two or more antibodies of thesecond antibody set 624 binding to their proximal targets on the secondantigen.

In an aspect, the two or more antigens in the test sample are free insolution. In an aspect, the two or more antigens in the test sample arelocated on the surface of one or more cell types in the test sample. Forexample, the two or more antigens can be associated with blood derivedcells (e.g., two or more antigens in or on red blood cells, platelets,white blood cells, etc.). For example, the two or more antigens can beassociated with malignant cells (e.g., human epidermal growth factorreceptor type 2 (Her2/neu) and carcinoembryonic antigen).

In an aspect, at least the first antibody set 614 or the second antibodyset 624 comprises at least one polyclonal antibody. For example, eachantibody set can include a polyclonal antibody which by definitionincludes a collection of immunoglobulins which may react with differentepitopes (i.e., different targets) on the same antigen. In an aspect,the first antibody set 614 includes one or more first polyclonalantibodies specific for proximal targets on the first antigen and thesecond antibody set 624 includes one or more second polyclonalantibodies specific for proximal targets on the second antigen. In anaspect, at least the first antibody set 614 or the second antibody set624 comprises two or more monoclonal antibodies. For example, eachantibody set can include two or more monoclonal antibodies, each ofwhich binds to different epitopes on the same antigen. In an aspect, thefirst antibody set 614 includes two or more monoclonal antibodiesspecific for proximal targets on the first antigen and the secondantibody set 624 includes two or more monoclonal antibodies specific forproximal targets on the second antigen.

The two or more antibodies of the first antibody set 614 and of thesecond antibody set 624 include modifications capable of interacting inan antibody-based proximity assay. The modifications can include a pairof molecules, wherein a first molecule of the pair is associated withone of the two or more antibodies and the second molecule of the pair isassociated with a second of the two or more antibodies. When the firstmolecule and the second molecule come in contact with one another byvirtue of the respective antibodies binding to their targets, the firstmolecule and the second molecule are able to interact. In someembodiments, the modification to the antibodies is a covalentmodification. For example, the pair of molecules can be covalentlyattached to the antibodies through a cross-linking agent(s),non-limiting examples of which include NHS-ester, imidoester, maleimide,pyridyldithiol, hydrazide, and carbodiimide reactive groups (see, e.g.,Thermo Fisher Scientific for an extensive listing of cross-linkingagents). In some embodiments, the modification to the antibodies is anon-covalent modification. For example, the pair of molecules can benon-covalently attached to the antibodies through an avidin/biotinbinding interaction.

In an aspect, the two or more antibodies of the first antibody set 614and the two or more antibodies of the second antibody set 624 includeoligonucleotide modifications. For example, the modifications caninclude a pair of oligonucleotides, wherein a first oligonucleotide ofthe pair is associated with one of the two or more antibodies of a givenset of antibodies and the second oligonucleotide of the pair isassociated with a second of the two or more antibodies of that given setof antibodies. When the first oligonucleotide and the secondoligonucleotide come in contact with one another by virtue of therespective antibodies binding to their targets, the firstoligonucleotide and the second oligonucleotide are able to interact. Inan aspect, a first antibody of the two or more antibodies of an antibodyset is modified with a first oligonucleotide and a second antibody ofthe two or more antibodies of the antibody set is modified with a secondoligonucleotide, wherein the first oligonucleotide and the secondoligonucleotide are capable of interacting with one another when thefirst antibody and the second antibody bind their respective proximaltargets on an antigen. In an aspect, the oligonucleotide modificationsform a template for amplification when in close proximity In an aspect,the oligonucleotide modifications are capable of undergoing a ligationreaction when in close proximity For example, when a firstoligonucleotide associated with one of the two or more antibodies of anantibody set and a second oligonucleotide associated with a second ofthe two or more antibodies of the antibody set come into proximity toone another by virtue of the antibodies binding their respectiveproximal targets, a ligation assay can be performed to connect the firstand second oligonucleotides to form a single oligonucleotide for use asan amplification target. See, e.g., Greenwood, et al. (2015)Biomolecular Detection Quantification 4:10-16, which is incorporatedherein by reference. In this instance, the at least one opticallydetectable reporter probe can include a DNA intercalating dye, e.g.,EvaGreen, that can be used to detect formation of an amplificationproduct from the ligated oligonucleotide template.

In an aspect, the oligonucleotide modifications are capable ofhybridizing to one another when in close proximity For example, when afirst oligonucleotide associated with one of the two or more antibodiesof a given set of antibodies and a second oligonucleotide associatedwith a second of the two or more antibodies of the given set ofantibodies come into proximity to one another by virtue of theantibodies binding their respective proximal targets, at least a portionof the first and second oligonucleotides are capable of hybridizing toone another to create a double stranded DNA. The overlapped doublestranded DNA can be extended using standard amplification procedures.Amplification can be monitored by an optically detectable reporter probethat is a DNA intercalating dye, non-limiting examples of which havebeen described above herein.

In an aspect, the two or more antibodies of the first antibody set 614and the two or more antibodies of the second antibody set 624 include atleast one donor-acceptor pair. In an aspect, a donor molecule isincorporated into a first antibody of an antibody set and an acceptormolecule is incorporated into a second antibody of the antibody set.When the first antibody and the second antibody bind to proximal targetson the surface of a cell, the donor molecule and the acceptor moleculeare able to come into close contact and interact with one another. In anaspect, the donor molecule is capable of being excited by a stimulus(e.g., light, pH, temperature) causing a reaction (usually emission ofsome specific wavelength of light) to occur from the acceptor moleculeif the donor and the acceptor are in close proximity In an aspect, thedonor-acceptor pair includes a fluorophore-quencher pair, wherein afluorophore molecule is incorporated into a first antibody of anantibody set and a quencher molecule is incorporated into a secondantibody of the antibody set and upon binding of the first antibody andthe second antibody to their respectively proximal targets, thefluorophore is quenched. Non-limiting examples of donor-acceptor pairsand fluorophore-quencher pairs have been described above herein.

System 600 for multiplexed analysis of two or more antigens in a testsample includes a first set of particles 610 degradable in response to afirst environmental condition and at least one second set of particles620 degradable in response to a second environmental condition. In anaspect, the first environmental condition and the second environmentalcondition comprises at least one of temperature, pH, chemical reaction,electric field, or electromagnetic energy. For example, the first set ofparticles and the at least one second set of particles can be degradablein response to a change in at least one of temperature, pH, chemicalreaction, electric field, or electromagnetic energy. For example, thefirst set of particles and the at least one second set of particles canbe formed from materials that are degradable in response to a change inat least one of temperature, pH, chemical reaction, electric field, orelectromagnetic energy.

In some embodiments, the first environmental condition and the secondenvironmental condition are identical environmental conditions. Forexample, the first set of particles and the at least one second set ofparticles can be degradable in response to identical environmentalconditions, for example, an identical condition of temperature, pH,chemical reaction, electric field, and/or electromagnetic energy. Forexample, the first set of particles and the at least one second set ofparticles can be formed from materials that are degradable in responseto identical environmental conditions of temperature, pH, chemicalreaction, electric field, and/or electromagnetic energy.

In an aspect, the particles of the first set of particles 610 and of theat least one second set of particles 620 include a structure formed froma degradable material that is degradable in response to at least thefirst environmental condition or the second environmental condition. Inan aspect, the particles of the first set of particles 610 and of the atleast one second set of particles 620 include a structure formed fromlow melt agarose. In an aspect, the particles of the first set ofparticles 610 and of the at least one second set of particles 620include a structure formed from at least one of a degradable gel, adegradable alginate, or a degradable sugar. In an aspect, the particlesof the first set of particles 610 and of the at least one second set ofparticles 620 include a structure formed from a temperature-responsivematerial. In an aspect, the particles of the first set of particles 610and of the at least one second set of particles 620 include a structureformed from at least one of a pH-responsive degradable material, achemically-responsive degradable material, an electric field-responsivedegradable material, or an electromagnetic energy-responsive degradablematerial. Non-limiting examples of environmental condition-responsivedegradable materials for forming the particles of the two or more setsof particles are presented above herein.

The two or more sets of particles 605 include components for multiplexanalysis two or more antigens. In some embodiments, the multiplexanalysis of two or more antigens includes performing reactions inaqueous-in-oil droplets or an emulsion system. In an aspect, at least aportion of the particles of the first set of particles 610 and of the atleast one second set of particles 620 are distributable into an aqueousportion of aqueous-in-oil droplets or emulsion. In an aspect, theparticles of the first set of particles 610 and of the at least onesecond set of particles 620 are hydrophilic. In an aspect, the particlesof the first set of particles 610 and of the at least one second set ofparticles 620 include a structure at least partially formed from ahydrophilic material. Non-limiting examples of hydrophilic material foruse in forming hydrophilic degradable particles have been describedabove herein.

System 600 for multiplexed analysis of two or more antigens in a testsample includes a first set of particles 610 including a first opticallydetectable identifier 616 capable of emitting a first wavelength, atleast one second set of particles 620 including a second opticallydetectable identifier 626 capable of emitting a second wavelength, andat least one optically detectable reporter probe 630 capable ofconstitutively emitting a third wavelength. In an aspect, the firstwavelength emitted by the first optically detectable identifier 616 is afirst detectable color, the second wavelength emitted by the secondoptically detectable identifier 626 is a second detectable color, andthe third wavelength constitutively emitted by the at least oneoptically detectable reporter probe 630 is a third detectable color. Inan aspect, the first wavelength, the second wavelength, and the thirdwavelength are optically discernable from one another. In an aspect, thefirst wavelength, the second wavelength, or the third wavelength is anultraviolet wavelength or wavelength band of electromagnetic energy. Inan aspect, at least one of the first wavelength, the second wavelength,or the third wavelength is a visible wavelength or wavelength band ofelectromagnetic energy. In an aspect, the first wavelength, the secondwavelength, or the third wavelength is a near infrared wavelength orwavelength band of electromagnetic energy.

In an aspect, the first optically detectable identifier 616 is a firstcolored dye or pigment and the second optically detectable identifier626 is a second colored dye or pigment. In an aspect, the firstoptically detectable identifier 616 includes a first fluorophore capableof emitting fluorescence at the first wavelength and the secondoptically detectable identifier 626 includes a second fluorophorecapable of emitting fluorescence at the second wavelength. In an aspect,the first optically detectable identifier 616 is a first quantum dotcapable of emitting the first wavelength and the second opticallydetectable identifier 626 is a second quantum dot capable of emittingthe second wavelength. Non-limiting examples of dyes, pigments,fluorophores, colored or fluorescing particles, or quantum dots havebeen described above.

System 600 for multiplexed analysis of two or more antigens in a testsample includes at least one optically detectable reporter probe 630capable of constitutively emitting a third wavelength in response to thetwo or more antibodies of the first antibody set 614 binding to theirproximal targets on the first antigen and/or the two or more antibodiesof the second antibody set 624 binding to their proximal targets on thesecond antigen. In some embodiments, the at least one opticallydetectable reporter probe 630 is associated with each particle of thefirst set of particles 610 and with each particle of the at least onesecond set of particles 620. In some embodiments, the at least oneoptically detectable reporter probe 630 includes a first opticallydetectable reporter probe for specifically signaling in response to thetwo or more antibodies of the first antibody set 614 binding theirproximal targets on the first antigen and a second optically detectablereporter probe for specifically signaling in response to the two or moreantibodies of the second antibody set 624 binding their proximal targetson the second antigen. For example, a first optically detectablereporter probe can include a first donor-acceptor pair modification tothe two or more antibodies associated with the first set of antibodies614 and a second optically detectable reporter probe can include asecond donor-acceptor pair modification to the two or more antibodiesassociated with the second set of antibodies 624. In some instance, thefirst donor-acceptor pair of the first antibody set and the seconddonor-acceptor pair of the second antibody set are the same, emittingthe same third wavelength in response to respective binding of the firstantibody set to proximal targets on the first antigen and binding of thesecond antibody set to proximal targets on the second antigen. In thisinstance, the two different reporter probes constitutively emit the samewavelength indicative of antibody binding while the optically detectableidentifier provides an indication of which antigen is present.

In an aspect, the at least one optically detectable reporter probe 630comprises a donor-acceptor pair capable of constitutively emitting thethird wavelength. In an aspect, a donor molecule of a donor-acceptorpair is incorporated into a portion of a first antibody and an acceptormolecule of the donor-acceptor pair is incorporated into a secondantibody and upon binding of the first antibody and the second antibodyto their respectively proximal targets, the donor is excited by astimulus (e.g., light, pH, temperature) causing a reaction (usuallyemission of some specific wavelength of light) to occur from theacceptor if the donor and the acceptor are in close proximity

In an aspect, the at least one optically detectable reporter probe 630is capable of constitutively emitting the third wavelength in responseto an amplification reaction. For example, the optically detectablereporter probe can detect amplification of oligonucleotide modificationsto the two or more antibodies of an antibody set. Non limiting examplesof optically detectable reporter probes for detecting amplification havebeen describe above herein. In an aspect, the at least one opticallydetectable reporter probe 630 includes a TaqMan-like probe capable ofconstitutively emitting the third wavelength. For example, the at leastone optically detectable reporter probe can include a TaqMan probeconfigured to interact with the amplified nucleic acid sequencepresented by the interaction of oligonucleotides on antibodies bound toproximal targets on a cell surface. In an aspect, the at least oneoptically detectable reporter probe 630 comprises a DNA intercalatingagent capable of constitutively emitting the third wavelength. Forexample, the at least one optically detectable reporter probe caninclude EvaGreen or other intercalating dye capable of emitting awavelength in response to binding to double stranded DNA.

In an aspect, system 600 for multiplexed analysis of two or moreantigens in a test sample includes more than two sets of particles. Inan aspect, system 600 includes three, four, five, six, seven, eight,nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,seventeen, eighteen, nineteen, or twenty sets of particles, each set ofparticles includes an antibody set and an optically detectableidentifier.

In an aspect, system 600 includes at least one third set of particles,each particle of the at least one third set of particles degradable inresponse to at least one of the first, the second, or a thirdenvironmental condition and having associated therewith a third antibodyset and a third optically detectable identifier capable of emitting afourth wavelength, the third antibody set including two or moreantibodies specific for proximal targets on a third antigen, wherein thetwo or more antibodies of the third antibody set include modificationscapable of interacting in an antibody-based proximity assay, and thethird optically detectable identifier indicative of the third antigen;and wherein the at least one optically detectable reporter probe iscapable of constitutively emitting the third wavelength in response tothe two or more antibodies of the first antibody set binding to theirproximal targets on the first antigen, the two or more antibodies of thesecond antibody set binding to their proximal targets on the secondantigen, and/or the two or more antibodies of the third antibody setbinding to their proximal targets on the third antigen.

In some embodiments, system 600 is configured for multiplexed analysisof two or more antigens on a surface of two or more cell types. In anaspect, each particle of the first set of particles 610 is degradable inresponse to the first environmental condition and has associatedtherewith a first antibody set including two or more antibodies specificfor proximal targets on a surface of a first cell type, wherein the twoor more antibodies of the first antibody set include modificationscapable of interacting in an antibody-based proximity assay, and whereinthe first optically detectable identifier capable of emitting the firstwavelength is indicative of the first antibody set; wherein eachparticle of the at least one second set of particles 620 is degradablein response to the second environmental condition and has associatedtherewith a second antibody set including two or more antibodiesspecific for proximal targets on a surface of a second cell type,wherein the two or more second antibodies of the second antibody setinclude modifications capable of interacting in an antibody-basedproximity assay, and wherein the second optically detectable identifiercapable of emitting the second wavelength is indicative of the secondantibody set; and wherein the at least one optically detectable reporterprobe 630 is capable of constitutively emitting the third wavelength inresponse to the two or more antibodies of the first antibody set bindingto their proximal targets on the surface of the first cell type and/orthe two or more antibodies of the second antibody set binding to theirproximal targets on the surface of the second cell type.

In an aspect, the two or more antigens in the test sample are located onthe surface of one or more cell types in the test sample. For example,the two or more antigens can be associated with blood derived cells(e.g., red blood cells, platelets, white blood cells, etc.). In anaspect, the first cell type and the second cell type are immune celltypes. For example, the first antibody set and the second antibody setcan be selected or designed to bind antigens on the surface of immunecells. Non-limiting examples of immune cells include granulocytes (e.g.,basophils, eosinophils, and neutrophils), mast cells, monocytes,macrophages, dendritic cells, lymphocytes (e.g., B cells and T cells),and NK cells. For example, the two or more antigens can be associatedwith malignant cells (e.g., human epidermal growth factor receptor type2 (Her2/neu) and carcinoembryonic antigen).

In an aspect, the two or more antibodies of the first antibody set bindto the same antigen on the surface of the first cell type and the two ormore antibodies of the second antibody set bind to the same antigen onthe second cell type. For example, the two or more antibodies can bedesigned to bind different epitopes on the same antigen. In an aspect,the two or more antibodies of the first antibody set bind to differentantigens that are proximal to one another on the surface of the firstcell type and the two or more antibodies of the second antibody set bindto different antigens that are proximal to one another on the surface ofthe second cell type. For example, a subset of the two or moreantibodies can be designed to bind one target on a cell, e.g., T-cellreceptor, and a second subset of the two or more antibodies can bedesigned to bind a second proximal target on a cell, e.g., CD3, whereinthe T-cell receptor and CD3 form a complex on the cell membrane inresponse to T-cell activation.

In some embodiments, a kit may be provided, containing one or more ofthe above composition. A “kit,” as used herein, typically defines apackage or an assembly including one or more of the compositions of theinvention, and/or other compositions associated with the invention, forexample, as previously described. In some embodiments, each kit includestwo or more sets of particles and at least one optically detectablereporter probe, non-limiting aspects of which have been describedherein. For example, a kit may include two or more sets of particles andat least one optically detectable reporter probe for multiplexedanalysis of two or more targets in a test sample. For example, a kit mayinclude two or more sets of particles and at least one opticallydetectable reporter probe for multiplexed analysis of two or morenucleic acid sequences in a test sample. For example, a kit may includetwo or more sets of particles and at least one optically detectablereporter probe for multiplexed analysis of antibiotic resistance in abacterial sample. For example, a kit may include two or more sets ofparticles and at least one optically detectable reporter probe formultiplexed analysis of two or more antigens in a test sample.

In other embodiments, multiple kits may be combined to perform amultiplexed analysis. For example, a first kit may include at least onefirst set of particles and the at least one optically detectablereporter probe and at least one second kit may include at least onesecond set of particles and the at least one optically detectablereporter probe, wherein the first kit and the at least one second kitare combined to perform multiplexed analysis of two or more targets in asample. In this way, kits containing specific sets of particles can bemixed and matched as appropriate for the multiplexed analysis.

Each composition of a kit, e.g., each set of particles and at least oneoptically detectable reporter probe, can be proved in liquid form (e.g.,in solution), in solid form (e.g., a dried powder), etc. A kit mayfurther include one or more emulsifiers, e.g., mineral oil, and/orsurfactants for use in forming reaction droplets. A kit may furtherinclude other compositions, e.g., one or more reaction reagents,suitable for performing a reaction with the set of particles and the atleast one optically detectable reporter probe. For example, the kit caninclude reaction reagents necessary to perform an amplificationreaction. For example, the kit can include reaction reagents necessaryto culture cells, e.g., mammalian cells or bacteria. A kit may furtherinclude instructions. For example, the kit may include instructions forthe use, modification, mixing, diluting, preserving, administering,assembly, storage, packaging, and/or preparations of the compositionsand/or other compositions associated with the kit. The instructions maybe provided in any form recognizable by one of ordinary skill in the artas a suitable means for obtaining such instructions, e.g., written orpublished, verbal, audible, digital, optical, visual, or electroniccommunication (e.g., via the Internet or web-based communication).

Methods are described herein for multiplexed analysis. FIG. 7 provides ablock diagram of a non-limiting example of a method 700 for multiplexedanalysis of two or more targets in a test sample. Method 700 includes inblock 710 combining in an aqueous medium the test sample, one or morereaction reagents, a first set of particles, at least one second set ofparticles, and at least one optically detectable reporter probe, whereineach particle of the first set of particles is degradable in response toa first environmental condition and has associated therewith a first setof one or more target-specific reagents specific for a first target anda first optically detectable identifier capable of emitting a firstwavelength indicative of the first set of one or more target-specificreagents, wherein each particle of the at least one second set ofparticles is degradable in response to a second environmental conditionand has associated therewith a second set of one or more target-specificreagents specific for a second target and a second optically detectableidentifier emitting a second wavelength indicative of the second set ofone or more target-specific reagents, and wherein the at least oneoptically detectable reporter probe constitutively emits a thirdwavelength in response to reaction of the first set of one or moretarget-specific reagents with the first target in the test sample and/orto reaction of the second set of one or more target-specific reagentswith the second target in the test sample; in block 720, forming aplurality of reaction droplets by adding an immiscible carrier fluid tothe aqueous medium; in block 730 performing a reaction with theplurality of formed reaction droplets; in block 740, interrogating atleast a portion of the plurality of formed reaction droplets foremission of the first wavelength indicative of the first set of one ormore target-specific reagents, the second wavelength indicative of thesecond set of one or more target-specific reagents, and the thirdwavelength indicative of constitutive emission from the at least oneoptically detectable reporter probe; in block 750 reporting a number ofthe formed reaction droplets emitting both the first wavelength and thethird wavelength; and in block 760 reporting a number of the formedreaction droplets emitting both the second wavelength and the thirdwavelength.

Method 700 provides steps for multiplexed analysis of two or moretargets in a test sample. The two or more targets can include two ormore specific targets in the test sample. The two or more targets caninclude two or more specific analytes in the test sample. For example,the two or more targets can include two or more specific DNA targets,RNA targets, protein targets, antigen targets, carbohydrate targets,lipid targets, and the like. For example, the two or more targets canfurther include oligonucleotides, peptides, receptors, cell surfacemarkers, small molecule compounds, organic compounds, inorganiccompounds. For example, the two or more targets can include two or moretargets (e.g., DNA, RNA, protein, carbohydrate, lipid, and the like)associated with a specific cell type(s) (e.g., blood, body fluid, ortissue cells, bacteria, fungi, parasites, and the like).

Method 700 includes combining in an aqueous medium the test sample, oneor more reaction reagents, a first set of particles, at least one secondset of particles, and at least one optically detectable reporter probe.In an aspect, the aqueous medium comprises water. In an aspect, theaqueous medium comprises a physiological saline solution. For example,the aqueous medium can include a 0.9% solution of sodium chloride inwater. In an aspect, the aqueous medium comprises a buffered solution.For example, the aqueous medium can include phosphate buffered saline(PBS). In an aspect, the aqueous medium comprises a cell culture medium.For example, the aqueous medium can include a cell culture mediumappropriate for culture and growth of isolated mammalian cells. Forexample, the aqueous medium can include a nutrient rich medium or broth(e.g., Luria broth) appropriate for culture and growth of bacteria. Forexample, the aqueous medium can include a culture broth appropriate forculture and growth of other microorganisms (e.g., yeast or other fungi).

Method 700 includes in block 710 combining the test sample with anaqueous medium. In an aspect, the test sample is a complex test sample,including many components. In an aspect, the test sample is a simple ordefined test sample, including just two or more components. In anaspect, the test sample includes at least a portion of a bodily fluid(e.g., blood, urine, lymph, sputum, cerebrospinal fluid, semen, saliva,synovial fluid, mucus, amniotic fluid, vaginal secretions, breast milk,bile, aqueous humor, gastric acid, pus, phlegm, feces, or other bodilyfluid or secretion), a tissue sample (e.g., a biopsy sample), or a atleast a portion of a swab sample (e.g., a swab sample taken from asurface of a body or body part. In some embodiments, the test sampleincludes an environmental sample (e.g., a sample of water, air, soil, asurface, food, beverage, medicine, and the like). In an aspect, the testsample has been processed in some manner prior to combining with theother reagents. For example, the test sample may be processed to extractDNA or RNA from the sample prior to combining with the other reagents.For example, the test sample may be processed to extract one or more ofproteins, peptides, lipids, carbohydrates, or other cellular componentsprior to combining with the other reagents. In an aspect, the testsample is a defined sample including a defined population of targets.For example, the test sample can be formulated to contain a definednumber and set of targets for use in the multiplexed analysis.

Method 700 includes in block 710 adding one or more reaction reagents tothe aqueous medium. In an aspect, the one or more reaction reagents arenecessary and/or sufficient for performing the multiplex analysis. In anaspect, the one or more reaction reagents include one or more reagentssuitable for performing an amplification reaction. For example, the oneor more reaction reagents can include one or more reagents forperforming polymerase chain reaction (PCR) amplification such as, e.g.,a DNA polymerase (e.g., Taq polymerase), deoxynucleoside triphosphatesor deoxynucleotide triphosphates (dNTPs), a buffer solution, bivalentcations (e.g., magnesium or manganese ions), and monovalent cations(e.g., potassium ions). In an aspect, the one or more reaction reagentscomprise one or more reagents suitable for performing isothermalamplification, non-limiting examples of which have been described aboveherein.

In an aspect, the one or more reaction reagents include one or morecomponents of a growth or culture medium. For example, the one or morereaction reagents can include components of a growth or culture mediumfor culturing cells and can be either synthetic or chemically defined ornon-synthetic or chemically undefined. The culture or growth medium caninclude components to support growth of microorganisms (e.g., bacteriaor fungi) or cells (tissue culture cells or primary cells) and mayinclude a carbon source (e.g., glucose), various salts, and a source ofamino acids and nitrogen. In some embodiments, the culture or growthmedium includes a selective medium for selective growth of a type(s) ofcell. In some embodiments, the culture or growth medium includes adifferential or indicator medium for detecting selective growth of atype(s) of cell. The one or more reaction reagents can include any of anumber of nutrients, amino acids, lipids, carbohydrates, sugars, andsalts necessary to promote growth of a given cell type(s).

In an aspect, the one or more reaction reagents include one or morereagents necessary or sufficient for performing a chemical reaction. Inan aspect, the one or more reaction reagents include one or morereagents necessary or sufficient for an enzymatic reaction. For example,the reaction reagents can include a ligase and other reagents necessaryto perform a ligation reaction. For example, the one or more reactionreagents can include enzymes, substrates, buffer, salts, ions, inorganicor organic co-factors, co-enzymes, and any other reagents necessary orsufficient for a given enzymatic reaction.

Method 700 further includes in block 720 forming a plurality of reactiondroplets by adding an immiscible carrier fluid to the aqueous medium.The potential targets in the test sample as well as the two or more setsof particles and the at least one optically detectable reporter probe inthe aqueous medium are stochastically distributed into the plurality ofreaction droplets. The immiscible carrier fluid can include an oil,e.g., non-limiting examples of which include mineral oil, dioctylphthalate (DOP), oleic acid, octamethyltrisiloxane (OMTS), fluorinatedoils, perfluorinated oils, fluorocarbon oils (e.g., FluorinertTM FC-40,from 3M Company, Minneapolis Minn.). In some embodiments, the immisciblecarrier fluid further includes a surfactant. For example, the immisciblecarrier fluid can include a fluorocarbon carrier oil with 0.01%-2% (v/v)of a surfactant. In an aspect, the surfactant may act as a detergent,wetting agent, emulsifier, foaming agent, and/or dispersant.Non-limiting examples of surfactants include Zonyl®FSO-100, ABIL®EM 90,Tween 20, Triton X-100, and SPAN 80.

In an aspect, method 700 includes forming the plurality of reactiondroplets through bulk emulsion. For example, the plurality of reactiondroplets can be formed by vigorously shaking the mixture of the aqueousmedium and the immiscible carrier fluid. In some embodiments, the bulkemulsion by shaking can be done manually, e.g., vigorous shaking byhand. In some embodiments, the bulk emulsion by shaking can be doneusing a machine (e.g., a vortex mixer or sonicator) with more specific“shaking” (e.g., vortexing or sonication).

In an aspect, method 700 includes forming the plurality of reactiondroplets through micro-emulsification or two-phase microfluidicflow-focusing. See, e.g., Gu, et al. (2011) Int. J. Mol. Sci.12(4):2572-2597, which is incorporated herein by reference. For example,droplets of the aqueous medium including the sample, reaction reagents,two or more sets of particles, and the at least one optically detectablereporter probe dispersed in the immiscible carrier fluid, e.g., oil, canbe formed using flow-focusing geometry. In an aspect, the plurality ofreaction droplets are formed using a T-junction device. For example, thereaction droplets can be formed by flowing the aqueous medium through amicrofluidic device by pumping or pressure and independently flowing inthe immiscible carrier fluid at right angles (e.g., through a Tjunction) to the flow of the aqueous medium to pinch off aqueousdroplets. In an aspect, the plurality of reaction droplets are formedusing a flow focusing device. For example, the reaction droplets can beformed by flowing the aqueous medium through a central channel of amicrofluidic device and flowing the immiscible carrier fluid throughchannels on both sides and at right angle to the flow of the aqueousmedium prior to passing through a small orifice in the flow channel tofocus and pinch off the droplets. Devices for generating droplet throughtwo-phase microfluidic flow-focusing are commercially available (e.g.,QX200 Droplet Generator from Bio-Rad Laboratories Inc., Hercules,Calif.; Model 1530 Monodisperse Droplet Generator from MSP Corporation,Shoreview, Minn.).

In an aspect, method 700 includes forming the plurality of reactiondroplets using a microfluidic chip, e.g., using SlipChip or similartechnology. See, e.g., Du et al. (2009) Lab Chip 9(16):2286-2292, whichis incorporated herein by reference.

Method 700 further includes in block 730 performing a reaction with theplurality of formed reaction droplets. Reactions can include chemical,enzymatic, or biochemical reactions, binding reactions, proliferationreactions, and inhibition reactions. In an aspect, the method includesperforming an amplification reaction. For example, the set of one ormore target-specific reagents associated with each particle of the twoor more sets of particles can include a set of amplification primers andthe reaction reagents can include reagents sufficient for performing theamplification reaction. In an aspect, the amplification reactioncomprises a polymerase chain reaction (PCR) amplification. In an aspect,the amplification reaction comprises an isothermal amplificationreaction. Non-limiting examples of isothermal amplification reactionsinclude recombinase polymerase amplification (RPA), loop-mediatedisothermal amplification (LAMP), nucleic acid sequence basedamplification (NASBA), helicase-dependent amplification (HAD), andnicking enzyme amplification reaction (NEAR).

Method 700 further includes in block 740 interrogating at least aportion of the plurality of formed reaction droplets for emission of atleast the first wavelength indicative of the first set of one or moretarget-specific reagents, the second wavelength indicative of the secondset of one or more target-specific reagents, and the third wavelengthindicative of constitutive emission from the at least one opticallydetectable reporter probe. In some embodiments, interrogating the atleast a portion of the plurality of reaction droplets includesinterrogating each of the plurality of reaction droplets. In otherembodiments, interrogating the at least a portion of the plurality ofreaction droplets includes interrogating a representative portion orstatistically significant portion of the plurality of reaction dropletsto allow for extrapolation of the number and/or percentage of reactiondroplets emitting the first, second, third wavelengths, and combinationsthereof.

In an aspect, method 700 includes interrogating at least a portion ofthe plurality of formed reaction droplets using multiple lasers, lightemitting diodes, and/or bandwidth filters to interrogate and report thereactions occurring on the interrogated portion of the reactiondroplets. In an aspect, interrogation is performed on a planar surface(e.g., multi-welled plate or microscope slide). For example, at least aportion of the reaction droplets can be interrogated using lightmicroscopy. For example, at least a portion of the reaction droplets canbe interrogated using fluorescence microscopy with various filter cubesto allow detection of reaction droplets fluorescing at at least one ofthe first wavelength, the second wavelength, the third wavelength, or acombination thereof. For example, a representative sample of thereaction droplets can be placed on a microscope slide or similar planarsubstrate and analyzed for a first wavelength of fluorescence (e.g.,red), a second wavelength of fluorescence (e.g., blue), and a thirdwavelength of fluorescence (e.g., green).

In an aspect, method 700 includes interrogating at least a portion ofthe plurality of formed reaction droplets using multiple lasers, lightemitting diodes, and/or bandwidth filters and high-speed digital-signalprocessing to report the reactions occurring on each individual reactiondroplet. For example, interrogating at least a portion of the pluralityof reaction droplets for emission of the first, second, and thirdwavelengths can be performed using a form of droplet reader thatincludes a means for interrogating emissions, e.g., fluorescence, fromreaction droplets. A non-limiting example of a droplet reader includesthe QX200 Droplet Reader (from Bio-Rad Laboratories, Hercules, Calif.).In an aspect, the method includes interrogating at least a portion ofthe plurality of reaction droplets for emission of the first, second,and third wavelengths using flow cytometry.

In an aspect, method 700 includes interrogating at least a portion ofthe plurality of formed reaction droplets with electromagnetic energy.For example, the reaction droplets can be interrogated by shining alight or using ambient light to observe one or more emitted wavelengths,e.g., color(s), from each of the at least a portion of the reactiondroplets. For example, the reaction droplets can be interrogated using alight microscope, either by manually counting the number of differentcolored reaction droplets or automatically by taking digital images ofmultiple fields and using signal processing to count the number ofdifferent colored reaction droplets.

In an aspect, method 700 includes directing a specific wavelength orwavelength band on the plurality of formed reaction droplets to elicitemission of a wavelength or wavelength band from the opticallydetectable identifiers and/or the optically detectable reporter probeassociated with the reaction droplets. For example, each of theplurality of reaction droplets can be interrogated with electromagneticenergy from a laser (e.g., helium-neon or argon lasers) to elicitemission of fluorescence (e.g., red, green, yellow, or bluefluorescence) from one or more of the optically detectable identifiersand/or the optically detectable reporter probe associated with thereaction droplets. A laser for use in interrogating the reactiondroplets can include a gas laser, a solid-state laser, a photoniccrystal laser, a semiconductor laser (e.g., laser diodes), or dyelasers.

Method 700 includes in block 750 and 760 reporting results frominterrogation of the at least a portion of the plurality of formedreaction droplets. Method 700 includes in block 750 reporting a numberof the formed reaction droplets emitting both the first wavelength andthe third wavelength. In an aspect, a reaction droplet emitting both thefirst wavelength and third wavelength can be classified as a positivedroplet. For a given reaction droplet, emitting the first wavelength isan indication that at least one particle from the first set of particlesis incorporated into the reaction droplet. That first wavelength iscorrelated with a first target. If that same reaction droplet emits thethird wavelength, this indicates that the desired reaction occurred,e.g., amplification, and the desired target, e.g., the first target, isin fact present in the test sample and was amplified. Method 700 furtherincludes in block 760 reporting a number of the formed reaction dropletsemitting both the second wavelength and the third wavelength. Dropletsemitting the second wavelength have at least one particle from the atleast one second set of particles incorporated into the reactiondroplet, wherein that second wavelength is correlated with a secondtarget. Again, if the same droplet emits both the second wavelength andthe third wavelength, it is an indication that the second target hasbeen identified in the sample. In an aspect, a reaction droplet emittingboth the second wavelength and third wavelength can be classified as apositive droplet indicative of the second target. In general, eithermanual counting or automated counting combined with signal processing isemployed to count the number and/or percentage of reaction dropletsemitting at least one of the first, second, and third wavelengths.

In an aspect, the method includes reporting the number of reactiondroplets emitting the first and third wavelengths and a number ofreaction droplets emitting the second and third wavelengths. Forexample, the reporting can include reporting a number of dropletsemitting both a red fluorescence and a blue fluorescence and a number ofdroplets emitting both a green fluorescence and a blue fluorescence. Inan aspect, reporting can include providing a percentage of reactiondroplets interrogated emitting the first and third wavelengths and apercentage of reaction droplets interrogated emitting the second andthird wavelengths. For example, the reporting can include reporting apercentage of droplets emitting both a red fluorescence and a bluefluorescence and a percentage of droplets emitting both a greenfluorescence and a blue fluorescence. In an aspect, reporting can benumerical. For example, the reporting can include a number or percentageof positive droplets. In an aspect, reporting can be graphical. Forexample, the reporting can include a graphical representation (e.g.,scatter blot, bar graph, etc.) representing of positive droplets.

In some embodiments, the method described in FIG. 7 is designed orconfigured for multiplexed analysis of two or more nucleic acidsequences in a test sample, such as illustrated in the block diagram ofFIG. 8 . FIG. 8 illustrates the steps of method 800 for multiplexedanalysis of two or more nucleic acid sequence targets in a test sample.Method 800 includes in block 810, combining in an aqueous medium thetest sample, one or more reaction reagents, a first set of particles, atleast one second set of particles, and at least one optically detectablereporter probe, wherein each particle of the first set of particles isdegradable in response to a first environmental condition and hasassociated therewith a first amplification primer set selected tointeract with a first nucleic acid sequence and a first opticallydetectable identifier capable of emitting a first wavelength indicativeof the first amplification primer set, wherein each particle of the atleast one second set of particles is degradable in response to a secondenvironmental condition and has associated therewith a secondamplification primer set selected to interact with a second nucleic acidsequence and a second optically detectable identifier emitting a secondwavelength indicative of the second amplification primer set, andwherein the at least one optically detectable reporter probe is capableof constitutively emitting a third wavelength in response toamplification of the first nucleic acid sequence and/or the secondnucleic acid sequence; in block 820, forming a plurality of reactiondroplets by adding an immiscible carrier fluid to the aqueous medium; inblock 830, performing an amplification reaction with the plurality offormed reaction droplets; in block 840, interrogating at least a portionof the plurality of formed reaction droplets for emission of the firstwavelength indicative of the first amplification primer set, the secondwavelength indicative of the second amplification primer set, and thethird wavelength indicative of constitutive emission from the at leastone optically detectable reporter probe in response to amplification ofthe first and/or the second nucleic acid sequence in the formed reactiondroplets; in block 850, reporting a number of the formed reactiondroplets emitting both the first wavelength and the third wavelength;and in block 860, reporting a number of the formed reaction dropletsemitting both the second wavelength and the third wavelength.

In some embodiments, the method described in FIG. 7 is designed orconfigured for multiplexed analysis of two or more bacterial nucleicacid sequences in a test sample. The method can include combining in anaqueous medium the test sample, one or more reaction reagents, a firstset of particles, at least one second set of particles, and at least onefluorescent intercalating agent, wherein each particle of the first setof particles is degradable in response to a first temperature conditionand has associated therewith a first amplification primer set selectedto interact with a first bacterial nucleic acid sequence and a firstfluorescent identifier capable of emitting a first wavelength indicativeof the first amplification primer set, wherein each particle of the atleast one second set of particles is degradable in response to a secondtemperature condition and has associated therewith a secondamplification primer set selected to interact with a second bacterialnucleic acid sequence and a second fluorescent identifier emitting asecond wavelength indicative of the second amplification primer set, andwherein the at least one fluorescent intercalating agent is capable ofconstitutively emitting a third wavelength in response to amplificationof the first bacterial nucleic acid sequence and/or the second bacterialnucleic acid sequence; forming a plurality of reaction droplets byadding an immiscible carrier fluid to the aqueous medium; performing anamplification reaction with the plurality of formed reaction droplets;interrogating at least a portion of the plurality of formed reactiondroplets for emission of the first wavelength indicative of the firstamplification primer set, the second wavelength indicative of the secondamplification primer set, and the third wavelength indicative ofconstitutive emission from the at least one fluorescent intercalatingagent in response to amplification of the first and/or the secondbacterial nucleic acid sequence in the formed reaction droplets;reporting a number of the formed reaction droplets emitting both thefirst wavelength and the third wavelength; and reporting a number of theformed reaction droplets emitting both the second wavelength and thethird wavelength.

In some embodiments, the method described in FIG. 7 is designed orconfigured for multiplexed analysis of antibiotic resistance in abacterial sample, such as illustrated in the block diagram of FIG. 9 .FIG. 9 illustrates the steps of method 900 for multiplexed analysis ofantibiotic resistance in a bacterial sample. Method 900 includes inblock 910, combining in an aqueous medium the bacterial sample, one ormore reaction reagents, a first set of particles, at least one secondset of particles, and at least one optically detectable reporter probe,wherein each particle of the first set of particles is degradable inresponse to a first environmental condition and has associated therewitha first antibiotic having at least one of bactericidal or bacteriostaticactivity against a first subset of bacteria and a first opticallydetectable identifier capable of emitting a first wavelength indicativeof the first antibiotic, wherein each particle of the at least onesecond set of particles is degradable in response to a secondenvironmental condition and has associated therewith a second antibiotichaving at least one of bactericidal or bacteriostatic activity against asecond subset of bacteria and a second optically detectable identifiercapable of emitting a second wavelength indicative of the secondantibiotic, and wherein the at least one optically detectable reporterprobe is capable of constitutively emitting a third wavelength inresponse to viability of bacteria in the bacterial sample; in block 920,forming a plurality of reaction droplets by adding an immiscible carrierfluid to the aqueous medium; in block 930, performing a reaction withthe plurality of formed reaction droplets; in block 940, interrogatingat least a portion of the plurality of formed reaction droplets foremission of the first wavelength indicative of the first antibiotic, thesecond wavelength indicative of the second antibiotic, and the thirdwavelength indicative of constitutive emission from the at least oneoptically detectable reporter probe in response to viability of bacteriain the bacterial sample; in block 950, reporting a number of the formedreaction droplets emitting both the first wavelength and the thirdwavelength; and in block 960, reporting a number of the formed reactiondroplets emitting both the second wavelength and the third wavelength.

In an aspect, method 900 includes performing a reaction with theplurality of formed reaction droplets including a bacterial sample andantibiotics. In some embodiments, the reaction includes a proliferationassay in which the bacteria in the bacterial sample are allowed toproliferate or not depending upon their susceptibility to theantibiotics present in the multiplex assay.

In some embodiments, the method described in FIG. 7 is designed orconfigured for multiplexed analysis of two or more antigens in a testsample, such as illustrated in the block diagram of FIG. 10 . FIG. 10illustrates the steps of method 1000 for multiplexed analysis of two ormore antigens in a test sample. Method 1000 includes in block 1010,combining in an aqueous medium the test sample, one or more reactionreagents, a first set of particles, at least one second set ofparticles, and at least one optically detectable reporter probe, whereineach particle of the first set of particles is degradable in response toa first environmental condition and has associated therewith a firstantibody set and a first optically detectable identifier capable ofemitting a first wavelength, the first antibody set including two ormore antibodies specific for proximal targets on a first antigen, thetwo or more antibodies of the first antibody set including modificationscapable of interacting in an antibody-based proximity assay, and thefirst optically detectable identifier indicative of the first antibodyset, wherein each particle of the second set of particles is degradablein response to a second environmental condition and has associatedtherewith a second antibody set and a second optically detectableidentifier capable of emitting a second wavelength, the second antibodyset including two or more antibodies specific for proximal targets on asecond antigen, the two or more antibodies of the second antibody setincluding modifications capable of interacting in an antibody-basedproximity assay, and the second optically detectable identifierindicative of the second antibody set, and wherein the at least oneoptically detectable reporter probe is capable of constitutivelyemitting a third wavelength in response to the two or more antibodies ofthe first antibody set binding to their proximal targets on the firstantigen and/or the two or more antibodies of the second antibody setbinding to their proximal targets on the second antigen; in block 1020,forming a plurality of reaction droplets by adding an immiscible carrierfluid to the aqueous medium; in block 1030, performing a reaction withthe plurality of formed reaction droplets; in block 1040, interrogatingat least a portion of the plurality of formed reaction droplets foremission of the first wavelength indicative of the first antibody set,the second wavelength indicative of the second antibody set, and thethird wavelength indicative of constitutive emission from the at leastone optically detectable reporter probe in response to the two or moreantibodies of the first antibody set binding to their proximal targetson the first antigen and/or the two or more antibodies of the secondantibody set binding to their proximal targets on the second antigen; inblock 1050, reporting a number of the formed reaction droplets emittingboth the first wavelength and the third wavelength; and in block 1060,reporting a number of the formed reaction droplets emitting both thesecond wavelength and the third wavelength.

In an aspect, method 1000 includes performing a reaction with theplurality of formed reaction droplets including a test sample and setsof modified antibodies. In some embodiments, method 1000 includesperforming an antibody-based proximity assay. For example, the reactioncan include binding sets of antibodies modified with donor-acceptorpairs to their respective targets and measuring a signal from thedonor-acceptor pair. In some embodiments, method 1000 includesperforming an amplification reaction. For example, the reaction caninclude amplifying a template formed by hybridization ofoligonucleotides associated with the antibody sets. In some embodiments,method 1000 includes performing a ligation reaction followed by anamplification reaction. For example, the reaction can include ligatingthe ends of an oligonucleotide pair associated with the antibody settogether to form a template for amplification, e.g., PCR amplificationor isothermal amplification.

EXAMPLE 1 Forming Agarose Particles with Reagents

Described is a method for forming agarose particles includingfluorescent FluoSpheres.

A 0.50% solution of agarose for forming the particles was generated asfollows: 275 mg of low melt agarose (Sigma A9414) was added to 50 mL ofwater and heated in a microwave for 1 minute to dissolve the low meltagarose. 3.6 mL of the agarose solution was combined with 400 uL ofFluoSphere F8801 (red fluorescing 580/605 nm; from Thermo FisherScientific, Waltham, Mass.) in a 4 mL glass vial. The solution ofagarose/FluoSpheres was kept warm.

A mineral oil solution with 3% SPAN 80 was generated as follows: 120 uLof SPAN 80 surfactant was added to 3.88 mL of mineral oil and vigorouslyshaken to ensure proper mixing of the surfactant in the mineral oil.

Agarose particles were formed as follows using a Dolomite microfluidicssystem (Dolomite Microfluidics, Norwell, Mass.):

A remote chamber was heated on a hot plate at 130° C. Pumps for pumpingmineral oil solution and the heated agarose solution were primed andconnected to a microfluidics chip. The system was primed by flowing oilthrough the chip at 500 mbar. Agarose was then flowed into system andpressure adjusted to allow for formation of agarose droplets. The formedagarose particles were collected and stored in oil at 4° C.

To remove oil from the agarose particles, the agarose particles werediluted 1:50 in water, placed in an Amicon Ultra-0.5 Centrifugal FilterDevice, and centrifuged at 14,000×g for 5 min. Filtration was repeatedtwice more with 1:50 dilution in water.

FIGS. 11A-11D show the formed agarose particles and the fluorescenceassociated with the FluoSphere F8801. FIG. 11A shows a brightfieldmicroscope image at 200× magnification of formed agarose particles 1100in mineral oil/SPAN 80. FIG. 11B shows a composite image of thebrightfield image of FIG. 11A overlaid with a fluorescent image of thesame field. In this grayscale image, the red fluorescence from theFluoSphere F8801 is seen as bright dots 1110 overlaid with the agaroseparticles 1100. FIG. 11C shows a brightfield microscope image at 200×magnification of formed agarose particles 1100 after multiple washingsin water. FIG. 11D shows a composite image of the brightfield image ofFIG. 11C overlaid with a fluorescent image of the same field. In thisgrayscale image, the red fluorescence from the FluoSphere F8801 is seenas bright dots 1110.

EXAMPLE 2 Forming Reaction Droplets with Quantum Dots

Low melt agarose particles were formed as described above. CdSeS/ZnSalloyed quantum dots with fluorescence emission at 630 nm were added tothe agarose during particle formation. FIG. 12 shows a fluorescentmicroscope image at 200× magnification of the formed agarose particles.The agarose particles are fluorescing due to the associated quantum dotsas is represented by particles 1200 a and 1200 b.

EXAMPLE 3 Amplification Reaction

An amplification reaction was performed using reaction droplets. A setof particles was generated with low melt agarose as described above. Theparticles included forward and reverse amplification primers for therodA gene of E. coli and CdSeS/ZnS alloyed quantum dots withfluorescence emission at 630 nm for the optically detectable identifier.The optically detectable reporter probe was a sequence specific probewith FAM fluorophore (fluorescence emission at 520; green) and BHQ1quencher. FIG. 13 shows a microscope image at 200× magnification of theformed reaction droplets post PCR amplification. The bright reactiondroplet 1300 is positive for amplification (i.e., has detectablefluorescence attributable to un-quenching of the FAM fluorophore),suggesting that the imaged reaction droplet 1300 included the target ofinterest and at least one agarose particle including the amplificationprimers.

EXAMPLE 4 Forming Multiple Sets of Particles with Amplification Primers

A system for performing multiplexed PCR reactions to detect DNA fromfour different bacteria associated with sepsis in a sample includes atleast the following: a first set of particles, a second set ofparticles, a third set of particles, a fourth set of particles and areporter probe.

A first set of particles specific for Staphylococcus aureus bacteria isformed as follows. A solution including 500 nM each of forward andreverse primers specific to Staph ST228 gene and FluoSphere F8801 (redfluorescing 580/605 nm; from Thermo Fisher Scientific, Waltham, Mass.)in 0.5% to 3.0% low melt agarose in water at 65° C. is flowed throughthe center channel of a heated microfluidic device and focused bymineral oil/1% SPAN 80 (v/v) through a 14 micron nozzle to producedroplets ranging in size from 5 to 30 microns in diameter. The resultingfirst set of droplets are allowed to solidify by cooling for 30 minutes(and rinsed with X prior to storage in X or Y?).

A second set of particles specific for Staphylococcus aureus that ismethicillin resistant (MRSA) bacteria is formed as follows. A solutionincluding 500 nM each of forward and reverse primers specific to StaphmecA gene which indicates resistance to first line antibiotics andFluoSphere F8797 (blue fluorescing 350/440 nm; from Thermo FisherScientific) in 0.5% to 3.0% low melt agarose in water at 65° C. isflowed through the center channel of the heated microfluidic device andfocused by mineral oil/1% SPAN 80 (v/v), cooled, and stored as describedabove with regard to the first set of particles.

A third set of particles specific for E. coli bacteria is formed asfollows. A solution including 500 nM forward and reverse primersspecific to E. coli rodA gene and FluoSphere F8800 (orange fluorescing540/560 nm; from Thermo Fisher Scientific) in 0.5% to 3.0% low meltagarose in water at 65° C. is flowed through the center channel of theheated microfluidic device and focused by mineral oil/1% SPAN 80 (v/v),cooled, and stored as described above with regard to the first set ofparticles.

A fourth set of particles specific for Streptococcus pneumoniae bacteriais formed as follows. A solution including 500 nM forward and reverseprimers specific to Strep lytA gene and FluoSphere F8789 (dark redfluorescing; 660/680 nm; from Thermo Fisher Scientific) in 0.5% to 3.0%low melt agarose in water at 65° C. is flowed through the center channelof the heated microfluidic device and focused by mineral oil/1% SPAN 80(v/v) cooled, and stored as described above with regard to the first setof particles.

The reporter probe is EvaGreen (green fluorescing (498/533 nm; fromBiotium, Inc., Fremont Calif.). In some embodiments, the EvaGreen isincorporated into each of the four set of particles during the formationof each set of agarose particles. In some embodiments, the EvaGreen isadded during the stochastic formation of PCR reaction droplets (seebelow).

EXAMPLE 5 Isolating DNA

DNA is isolated from a blood sample of an individual diagnosed withsepsis using standard procedures. Briefly, a small quantity of blood(e.g., 200 ul) is extracted by finger prick from the subject. Acommercially available DNA extraction kit (e.g., QIAamp DNA Blood (fromQIAGEN, North American Headquarters, Germantown, Md.) or a similar kit)can be used to extract DNA from the blood by treating the sample withprotease or proteinase K, binding DNA to a spin column, washing, andeluting. The blood sample may be pre-treated with an additional lysisreagent (e.g., lysozyme (20 mg/ml) and/or lysostaphin (200 ug/ml))adequate for lysing Gram-positive and difficult to lyse bacteria.

EXAMPLE 6 Forming Reaction Droplets and Performing Amplification

Method of determining presence of bacterial DNA in sepsis sample withthe four sets of particles includes at least the steps as follows. Anaqueous solution including a plurality of each of the four sets ofparticles is mixed with the genomic DNA extracted from blood, thereporter probe EvaGreen and a PCR master mix including a DNA polymerase(e.g., Taq DNA Polymerase), deoxyribonucleotide triphosphates (e.g.,dATP, dTTP, dGTP, and dCTP), and magnesium chloride in a bufferedsolution. In some embodiments, a PCR master mix including EvaGreen maybe used for the purpose (from, e.g., Biotium).

Reaction droplets including the above components are generated asfollows. The aqueous solution described above is added in a 1:2 ratio toan oil/surfactant mixture (e.g., mineral oil/1% SPAN 80 (v/v)) to a 1.7mL Eppendorf tube. These immiscible fluids are vortexed with a vortexmixer (e.g., VWR analog vortex mixer (cat. #10153-838; Radnor, Pa., USA)at a maximum speed for 10-20 seconds to create a population ofpolydisperse droplets. Alternatively, the reaction droplets are formedusing the above described aqueous solution in an automated dropletgenerator (from, e.g., BioRad, Hercules, Calif., USA) using amicrofluidic chip and focusing with an oil/surfactant mixture.

The reaction droplets are then run in a thermocycler (e.g., a C100 orC1000 thermocycler from BioRad, Hercules, Calif.) using the followingprotocol: 50° C. hold for 60 minutes, 95° C. hold for 10 minutes, 40cycles of 95° C. for 30 seconds and 60° C. for 1 minute. Fluorescenceassociated with fluorescent spheres and the EvaGreen is read using afluorescent reader (e.g., QX200 Droplet Reader, BioRad) and appropriatesoftware.

EXAMPLE 7 Multiplexed Analysis of Bacteria in a Test Sample

Described is an example of a system for multiplexed analysis of bacteriain a test sample of water. The system includes a set of particlesincluding the enzymatic substrate 4-methylumbelliferyl-β-D-glucuronide(MUG) for detecting enterobacteria (e.g., E. coli) and the generic cellgrowth marker resazurin. Methylumbelliferyl-β-D-glucuronide fluorescesblue in response to hydrolysis by β-D-glucuronide. Resazurin is acell-permeable and non-toxic oxidation-reduction indicator which inviable cells is reduced to red fluorescing resorufin. Particlescontaining methylumbelliferyl-β-D-glucuronide and resazurin (both from,e.g., Sigma-Aldrich, St. Louis, Mo.) are mixed into 0.5% to 3.0% lowmelt agarose in water at 65° C. and flowed through a center channel of aheated microfluidic device and focused by mineral oil/1% SPAN 80 (v/v),cooled, and stored.

The test sample of water is mixed with the set of particles as well as anutrient broth (e.g., LB broth). The aqueous solution is mixed with animmiscible carrier fluid, e.g., mineral oil to form reaction droplets.Droplets are formed with bacteria as described in Byrnes et al. Analyst(2018) 143:2828-2836, which is incorporated herein by reference. Thedroplets can be formed either by bulk emulsion using a vortex mixer orby flow-focusing using a microfluidic device as described above herein.

EXAMPLE 8 Multiplexed Immunoassay Detection

Described is an example of a system for multiplexed immunoassaydetection combined with DNA amplification. A first set of particlesincludes a polyclonal antibody specific for IL-8 protein. A second setof particles includes a polyclonal antibody specific for IL-12 protein.Polyclonal antibodies for both IL-8 and IL-12 are available from avariety of commercial sources (see, Linscott's Directory on the WorldWide Web at linscottsdirectory.com for an extensive list of suppliers).Each of these polyclonal antibodies is reactive against multipleepitopes on the target proteins. The anti-IL-8 polyclonal antibody andthe anti-IL-12 polyclonal antibody are modified withsuccinimidyl-4-[p-maleimidopheynyl]butyrate (SMPB) (from, e.g.,Pierce/Thermo Fischer Scientific) using the manufacturer's instructions.A G-50 spin column is used to remove excess SMPB. An oligonucleotidepair, each modified with either a 5 prime —SH group or a 3 prime —SHgroup, are incubated with half each of the SMPB-modified anti-IL-8 andanti-IL-12 polyclonal antibodies to allow cross-linking of theoligonucleotides to the antibodies (see, e.g., Gullberg, et al. (2004)Proc. Natl. Acad. Sci. 101:8420-8424, which is incorporated herein byreference).

A first set of particles specific for IL-8 protein is formed as follows.A solution including the anti-IL-8 polyclonal antibodies modified withthe oligonucleotide pair and FluoSphere F8801 (red fluorescing 580/605nm; from Thermo Fisher Scientific, Waltham, Mass.) in 0.5% to 3.0% lowmelt agarose in water at 65° C. is flowed through the center channel ofa heated microfluidic device and focused by mineral oil/1% SPAN 80 (v/v)through a 14 micron nozzle to produce droplets ranging in size from 5 to30 microns in diameter. The resulting first set of droplets are allowedto solidify by cooling for 30 minutes.

A second set of particles specific for IL-12 protein is formed asfollows. A solution including the anti-IL-12 polyclonal antibodiesmodified with the oligonucleotide pair and FluoSphere F8797 (bluefluorescing 350/440 nm; from Thermo Fisher Scientific) in 0.5% to 3.0%low melt agarose in water at 65° C. is flowed through the center channelof the heated microfluidic device and focused by mineral oil/1% SPAN 80(v/v), and cooled as described above.

The reporter probe is EvaGreen (green fluorescing (498/533 nm; fromBiotium, Inc., Fremont Calif.). In some embodiments, the EvaGreen isincorporated into the particles during the formation of each set ofagarose particles. In some embodiments, the EvaGreen is added to theaqueous solution (see below).

The particles including the anti-IL-8 and anti-IL-12 polyclonalantibodies are mixed in an aqueous solution with EvaGreen, ligationreagents, amplification reagents, and a test sample including IL-8 andIL-12 proteins. The ligation/amplification reagents can include T4 DNAligase, a connector oligonucleotide, dNTPs, and DNA polymerase (see,e.g., Gullberg, et al. (2004) Proc. Natl. Acad. Sci. 101:8420-8424,which is incorporated herein by reference). Aqueous-in-oil droplets areformed as described above. Ligation is carried out at room temperaturefollowed by PCR amplification as described above. Fluorescenceassociated with FluoSphere 8801, FluoSphere 8797, and EvaGreen is readusing a fluorescence reader.

One skilled in the art will recognize that the herein describedcomponent, devices, objects, and the discussion accompanying them areused as examples for the sake of conceptual clarity and that variousconfiguration modifications are contemplated. Consequently, as usedherein, the specific exemplars set forth and the accompanying discussionare intended to be representative of their more general classes. Ingeneral, use of any specific exemplar is intended to be representativeof its class, and the non-inclusion of specific components, devices, andobjects should not be taken as limiting.

With respect to the use of substantially any plural and/or singularterms herein, the plural can be translated to the singular and/or fromthe singular to the plural as is appropriate to the context and/orapplication. The various singular/plural permutations are not expresslyset forth herein for sake of clarity.

In some instances, one or more components can be referred to herein as“configured to,” “configured by,” “configurable to,” “operable/operativeto,” “adapted/adaptable,” “able to,” “conformable/conformed to,” etc.Those skilled in the art will recognize that such terms (e.g.“configured to”) can generally encompass active-state components and/orinactive-state components and/or standby-state components, unlesscontext requires otherwise.

While particular aspects of the present subject matter described hereinhave been shown and described, changes and modifications can be madewithout departing from the subject matter described herein and itsbroader aspects and, therefore, the appended claims are to encompasswithin their scope all such changes and modifications as are within thetrue spirit and scope of the subject matter described herein. Terms usedherein, and especially in the appended claims (e.g., bodies of theappended claims) are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.). If a specific number of an introduced claim recitation isintended, such an intent will be explicitly recited in the claim, and inthe absence of such recitation no such intent is present. For example,as an aid to understanding, the following appended claims can containusage of the introductory phrases “at least one” and “one or more” tointroduce claim recitations. However, the use of such phrases should notbe construed to imply that the introduction of a claim recitation by theindefinite articles “a” or “an” limits any particular claim containingsuch introduced claim recitation to claims containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should typically be interpreted to mean “atleast one” or “one or more”); the same holds true for the use ofdefinite articles used to introduce claim recitations. In addition, evenif a specific number of an introduced claim recitation is explicitlyrecited, such recitation should typically be interpreted to mean atleast the recited number (e.g., the bare recitation of “tworecitations,” without other modifiers, typically means at least tworecitations, or two or more recitations). Furthermore, in thoseinstances where a convention analogous to “at least one of A, B, and C,etc.” is used, in general such a construction is intended in the senseone having skill in the art would understand the convention (e.g.,“ asystem having at least one of A, B, and C” would include but not belimited to systems that have A alone, B alone, C alone, A and Btogether, A and C together, B and C together, and/or A, B, and Ctogether, etc.). In those instances where a convention analogous to “atleast one of A, B, or C, etc.” is used, in general such a constructionis intended in the sense one having skill in the art would understandthe convention (e.g.,“a system having at least one of A, B, or C” wouldinclude but not be limited to systems that have A alone, B alone, Calone, A and B together, A and C together, B and C together, and/or A,B, and C together, etc.). Typically a disjunctive word and/or phrasepresenting two or more alternative terms, whether in the description,claims, or drawings, should be understood to contemplate thepossibilities of including one of the terms, either of the terms, orboth terms unless context dictates otherwise. For example, the phrase “Aor B” will be typically understood to include the possibilities of “A”or “B” or “A and B.”

Aspects of the subject matter described herein are set out in thefollowing numbered paragraphs:

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non-patent publications referred to in this specification and/orlisted in any Application Data Sheet, are incorporated herein byreference, to the extent not inconsistent herewith.

While various aspects and embodiments have been disclosed herein, otheraspects and embodiments will be apparent to those skilled in the art.The various aspects and embodiments disclosed herein are for purposes ofillustration and are not intended to be limiting, with the true scopeand spirit being indicated by the following claims.

1-167. (canceled)
 168. A method for multiplexed analysis of two or moretargets in a test sample comprising: combining in an aqueous medium thetest sample, one or more reaction reagents, a first set of particles, atleast one second set of particles, and at least one optically detectablereporter probe, wherein each particle of the first set of particles isdegradable in response to a first environmental condition and hasassociated therewith a first set of one or more target-specific reagentsspecific for a first target and a first optically detectable identifiercapable of emitting a first wavelength indicative of the first set ofone or more target-specific reagents, wherein each particle of the atleast one second set of particles is degradable in response to a secondenvironmental condition and has associated therewith a second set of oneor more target-specific reagents specific for a second target and asecond optically detectable identifier emitting a second wavelengthindicative of the second set of one or more target-specific reagents,and wherein the at least one optically detectable reporter probeconstitutively emits a third wavelength in response to reaction of thefirst set of one or more target-specific reagents with the first targetin the test sample and/or to reaction of the second set of one or moretarget-specific reagents with the second target in the test sample;forming a plurality of reaction droplets by adding an immiscible carrierfluid to the aqueous medium; performing a reaction with the plurality offormed reaction droplets; interrogating at least a portion of theplurality of formed reaction droplets for emission of the firstwavelength indicative of the first set of one or more target-specificreagents, the second wavelength indicative of the second set of one ormore target-specific reagents, and the third wavelength indicative ofconstitutive emission from the at least one optically detectablereporter probe; reporting a number of the formed reaction dropletsemitting both the first wavelength and the third wavelength; andreporting a number of the formed reaction droplets emitting both thesecond wavelength and the third wavelength.
 169. A method formultiplexed analysis of two or more nucleic acid sequence targets in atest sample comprising: combining in an aqueous medium the test sample,one or more reaction reagents, a first set of particles, at least onesecond set of particles, and at least one optically detectable reporterprobe, wherein each particle of the first set of particles is degradablein response to a first environmental condition and has associatedtherewith a first amplification primer set selected to interact with afirst nucleic acid sequence and a first optically detectable identifiercapable of emitting a first wavelength indicative of the firstamplification primer set, wherein each particle of the at least onesecond set of particles is degradable in response to a secondenvironmental condition and has associated therewith a secondamplification primer set selected to interact with a second nucleic acidsequence and a second optically detectable identifier emitting a secondwavelength indicative of the second amplification primer set, andwherein the at least one optically detectable reporter probe is capableof constitutively emitting a third wavelength in response toamplification of the first nucleic acid sequence and/or the secondnucleic acid sequence; forming a plurality of reaction droplets byadding an immiscible carrier fluid to the aqueous medium; performing anamplification reaction with the plurality of formed reaction droplets;interrogating at least a portion of the plurality of formed reactiondroplets for emission of the first wavelength indicative of the firstamplification primer set, the second wavelength indicative of the secondamplification primer set, and the third wavelength indicative ofconstitutive emission from the at least one optically detectablereporter probe in response to amplification of the first and/or thesecond nucleic acid sequence in the formed reaction droplets; reportinga number of the formed reaction droplets emitting both the firstwavelength and the third wavelength; and reporting a number of theformed reaction droplets emitting both the second wavelength and thethird wavelength.
 170. A method for multiplexed analysis of antibioticresistance in a bacterial sample, comprising: combining in an aqueousmedium the bacterial sample, one or more reaction reagents, a first setof particles, at least one second set of particles, and at least oneoptically detectable reporter probe, wherein each particle of the firstset of particles is degradable in response to a first environmentalcondition and has associated therewith a first antibiotic having atleast one of bactericidal or bacteriostatic activity against a firstsubset of bacteria and a first optically detectable identifier capableof emitting a first wavelength indicative of the first antibiotic,wherein each particle of the at least one second set of particles isdegradable in response to a second environmental condition and hasassociated therewith a second antibiotic having at least one ofbactericidal or bacteriostatic activity against a second subset ofbacteria and a second optically detectable identifier capable ofemitting a second wavelength indicative of the second antibiotic, andwherein the at least one optically detectable reporter probe is capableof constitutively emitting a third wavelength in response to viabilityof bacteria in the bacterial sample; forming a plurality of reactiondroplets by adding an immiscible carrier fluid to the aqueous medium;performing a reaction with the plurality of formed reaction droplets;interrogating at least a portion of the plurality of formed reactiondroplets for emission of the first wavelength indicative of the firstantibiotic, the second wavelength indicative of the second antibiotic,and the third wavelength indicative of constitutive emission from the atleast one optically detectable reporter probe in response to viabilityof bacteria in the bacterial sample; reporting a number of the formedreaction droplets emitting both the first wavelength and the thirdwavelength; and reporting a number of the formed reaction dropletsemitting both the second wavelength and the third wavelength.
 171. Amethod for multiplexed analysis of two or more cell types in a testsample, comprising: combining in an aqueous medium the test sample, oneor more reaction reagents, a first set of particles, at least one secondset of particles, and at least one optically detectable reporter probe,wherein each particle of the first set of particles is degradable inresponse to a first environmental condition and has associated therewitha first antibody set and a first optically detectable identifier capableof emitting a first wavelength, the first antibody set including two ormore antibodies specific for proximal targets on a first antigen, thetwo or more antibodies of the first antibody set including modificationscapable of interacting in an antibody-based proximity assay, and thefirst optically detectable identifier indicative of the first antibodyset, wherein each particle of the second set of particles is degradablein response to a second environmental condition and has associatedtherewith a second antibody set and a second optically detectableidentifier capable of emitting a second wavelength, the second antibodyset including two or more antibodies specific for proximal targets on asecond antigen, the two or more antibodies of the second antibody setincluding modifications capable of interacting in an antibody-basedproximity assay, and the second optically detectable identifierindicative of the second antibody set, and wherein the at least oneoptically detectable reporter probe is capable of constitutivelyemitting a third wavelength in response to the two or more antibodies ofthe first antibody set binding to their proximal targets on the firstantigen and/or the two or more antibodies of the second antibody setbinding to their proximal targets on the second antigen. forming aplurality of reaction droplets by adding an immiscible carrier fluid tothe aqueous medium; performing a reaction with the plurality of formedreaction droplets; interrogating at least a portion of the plurality offormed reaction droplets for emission of the first wavelength indicativeof the first antibody set, the second wavelength indicative of thesecond antibody set, and the third wavelength indicative of constitutiveemission from the at least one optically detectable reporter probe inresponse to the two or more antibodies of the first antibody set bindingto the first antigen and/or the two or more antibodies of the secondantibody set binding to the second antigen; reporting a number of theformed reaction droplets emitting both the first wavelength and thethird wavelength; and reporting a number of the formed reaction dropletsemitting both the second wavelength and the third wavelength.
 172. Themethod of claim 168, wherein the particles of the first set of particlesand of the at least one second set of particles include a structure atleast partially formed from a hydrophilic material and degradable inresponse to at least a first temperature condition or a secondtemperature condition, wherein at least a portion of the particles ofthe first set of particles and of the at least one second set ofparticles are distributable into an aqueous portion of an aqueous-in-oildroplet.
 173. The method of claim 168, wherein the first wavelengthemitted by the first optically detectable identifier is a firstdetectable color, the second wavelength emitted by the second opticallydetectable identifier is a second detectable color, and the thirdwavelength constitutively emitted by the at least one opticallydetectable reporter probe is a third detectable color; and wherein thefirst wavelength, the second wavelength, and the third wavelength areoptically discernable from one another.
 174. The method of claim 168,wherein the first optically detectable identifier includes a firstfluorophore capable of emitting fluorescence at the first wavelength andthe second optically detectable identifier includes a second fluorophorecapable of emitting fluorescence at the second wavelength; and whereinthe at least one optically detectable reporter probe comprises afluorescent DNA intercalating agent capable of constitutively emittingthe third wavelength.
 175. The method of claim 169, wherein the firstamplification primer set and the second amplification primer set areselected to specifically interact with at least one of single strandedDNA sequences, double stranded DNA sequences, or RNA sequences.
 176. Themethod of claim 169, wherein the particles of the first set of particlesand of the at least one second set of particles include a structure atleast partially formed from a hydrophilic material and degradable inresponse to at least a first temperature condition or a secondtemperature condition, wherein at least a portion of the particles ofthe first set of particles and of the at least one second set ofparticles are distributable into an aqueous portion of an aqueous-in-oildroplet.
 177. The method of claim 169, wherein the first wavelengthemitted by the first optically detectable identifier is a firstdetectable color, the second wavelength emitted by the second opticallydetectable identifier is a second detectable color, and the thirdwavelength constitutively emitted by the at least one opticallydetectable reporter probe is a third detectable color; and wherein thefirst wavelength, the second wavelength, and the third wavelength areoptically discernable from one another.
 178. The method of claim 169,wherein the first optically detectable identifier includes a firstfluorophore capable of emitting fluorescence at the first wavelength andthe second optically detectable identifier includes a second fluorophorecapable of emitting fluorescence at the second wavelength; and whereinthe at least one optically detectable reporter probe comprises afluorescent DNA intercalating agent capable of constitutively emittingthe third wavelength.
 179. The method of claim 170, wherein the firstsubset of bacteria or the second subset of bacteria includes one or morestrains of Escherichia coli.
 180. The method of claim 170, wherein thefirst subset of bacteria or the second subset of bacteria includesMycobacterium tuberculosis.
 181. The method of claim 170, wherein thefirst subset of bacteria and the second subset of bacteria are identicalsubsets of bacteria.
 182. The method of claim 170, wherein the firstsubset of bacteria or the second subset of bacteria includes one or moreof methicillin resistant Staphylococcus aureus, methicillin susceptibleStaphylococcus aureus, Escherichia coli, Streptococcus pneumonia,Pseudomonas aeruginosa, Staphylococcus epidermidis, Salmonella enterica,Klebsiella pneumonia, Streptococcus pyogenes, Acinetobacter baumannii,or Enterococcus faecalis.
 183. The method of claim 170, wherein thefirst subset of bacteria or the second subset of bacteria includes oneor more of Enterococcus faecium, Proteus mirabilis, Streptococcusagalactiae, Staphylococcus saprophyticus, Corynebacterium urealyticum,Serratia marcescens, Klebsiella oxytoca, or Actinobaculum schaalii. 184.The method of claim 171, wherein at least the first antibody set or thesecond antibody set comprises at least one polyclonal antibody.
 185. Themethod of claim 171, wherein at least the first antibody set or thesecond antibody set comprises two or more monoclonal antibodies. 186.The method of claim 171, wherein the two or more antibodies of the firstantibody set and the two or more antibodies of the second antibody setinclude oligonucleotide modifications.
 187. The method of claim 171,wherein the first wavelength emitted by the first optically detectableidentifier is a first detectable color, the second wavelength emitted bythe second optically detectable identifier is a second detectable color,and the third wavelength constitutively emitted by the at least oneoptically detectable reporter probe is a third detectable color; andwherein the first wavelength, the second wavelength, and the thirdwavelength are optically discernable from one another.
 188. The methodof claim 171, wherein the first optically detectable identifier includesa first fluorophore capable of emitting fluorescence at the firstwavelength and the second optically detectable identifier includes asecond fluorophore capable of emitting fluorescence at the secondwavelength; and wherein the at least one optically detectable reporterprobe comprises a fluorescent DNA intercalating agent capable ofconstitutively emitting the third wavelength.